Sample records for based rna extraction

  1. Analysis of current and alternative phenol based RNA extraction methodologies for cyanobacteria

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    Lindblad Peter


    Full Text Available Abstract Background The validity and reproducibility of gene expression studies depend on the quality of extracted RNA and the degree of genomic DNA contamination. Cyanobacteria are gram-negative prokaryotes that synthesize chlorophyll a and carry out photosynthetic water oxidation. These organisms possess an extended array of secondary metabolites that impair cell lysis, presenting particular challenges when it comes to nucleic acid isolation. Therefore, we used the NHM5 strain of Nostoc punctiforme ATCC 29133 to compare and improve existing phenol based chemistry and procedures for RNA extraction. Results With this work we identify and explore strategies for improved and lower cost high quality RNA isolation from cyanobacteria. All the methods studied are suitable for RNA isolation and its use for downstream applications. We analyse different Trizol based protocols, introduce procedural changes and describe an alternative RNA extraction solution. Conclusion It was possible to improve purity of isolated RNA by modifying protocol procedures. Further improvements, both in RNA purity and experimental cost, were achieved by using a new extraction solution, PGTX.

  2. Novel extraction strategy of ribosomal RNA and genomic DNA from cheese for PCR-based investigations. (United States)

    Bonaïti, Catherine; Parayre, Sandrine; Irlinger, Françoise


    Cheese microorganisms, such as bacteria and fungi, constitute a complex ecosystem that plays a central role in cheeses ripening. The molecular study of cheese microbial diversity and activity is essential but the extraction of high quality nucleic acid may be problematic: the cheese samples are characterised by a strong buffering capacity which negatively influenced the yield of the extracted rRNA. The objective of this study is to develop an effective method for the direct and simultaneous isolation of yeast and bacterial ribosomal RNA and genomic DNA from the same cheese samples. DNA isolation was based on a protocol used for nucleic acids isolation from anaerobic digestor, without preliminary washing step with the combined use of the action of chaotropic agent (acid guanidinium thiocyanate), detergents (SDS, N-lauroylsarcosine), chelating agent (EDTA) and a mechanical method (bead beating system). The DNA purification was carried out by two washing steps of phenol-chloroform. RNA was isolated successfully after the second acid extraction step by recovering it from the phenolic phase of the first acid extraction. The novel method yielded pure preparation of undegraded RNA accessible for reverse transcription-PCR. The extraction protocol of genomic DNA and rRNA was applicable to complex ecosystem of different cheese matrices.

  3. Automation of DNA and miRNA co-extraction for miRNA-based identification of human body fluids and tissues. (United States)

    Kulstein, Galina; Marienfeld, Ralf; Miltner, Erich; Wiegand, Peter


    In the last years, microRNA (miRNA) analysis came into focus in the field of forensic genetics. Yet, no standardized and recommendable protocols for co-isolation of miRNA and DNA from forensic relevant samples have been developed so far. Hence, this study evaluated the performance of an automated Maxwell® 16 System-based strategy (Promega) for co-extraction of DNA and miRNA from forensically relevant (blood and saliva) samples compared to (semi-)manual extraction methods. Three procedures were compared on the basis of recovered quantity of DNA and miRNA (as determined by real-time PCR and Bioanalyzer), miRNA profiling (shown by Cq values and extraction efficiency), STR profiles, duration, contamination risk and handling. All in all, the results highlight that the automated co-extraction procedure yielded the highest miRNA and DNA amounts from saliva and blood samples compared to both (semi-)manual protocols. Also, for aged and genuine samples of forensically relevant traces the miRNA and DNA yields were sufficient for subsequent downstream analysis. Furthermore, the strategy allows miRNA extraction only in cases where it is relevant to obtain additional information about the sample type. Besides, this system enables flexible sample throughput and labor-saving sample processing with reduced risk of cross-contamination.

  4. Extraction and Detection of mRNA from a Single K562 Cell Based on the Functionalized Superparamagnetic Nanoparticles

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    ZHU,Long-Zhang; YU,Ping-Guo; SHEN,He-Bai; JIA,Neng-Qin; LONG,De-Hong; ZHOU,Hai-Qing


    A novel and promising method was developed to extract mRNA from a single cell based on the functionalized superparamagnetic nanoparticles.The oligo(dT)-coupled magnetite nanobeads were synthesized by the reaction of oligo(dT) and thiol-modified γ-Fe2O3 nanoparticles.The single cell was isolated from the massive cultivation according to a semi-quantum approaching technique and then lysed before mRNA separation.The oligo(dT)-coupled magnetite nanobeads were added to the crude lysates and then magnetic separation was preformed to get mRNA.The mRNA amplification through a two-step RT-PCR method was achieved.The agarose gel electrophoresis of PCR products after amplification shows that mRNA could be extracted from a single cell successfully.

  5. Evaluation of DNA and RNA extraction methods. (United States)

    Edwin Shiaw, C S; Shiran, M S; Cheah, Y K; Tan, G C; Sabariah, A R


    This study was done to evaluate various DNA and RNA extractions from archival FFPE tissues. A total of 30 FFPE blocks from the years of 2004 to 2006 were assessed with each modified and adapted method. Extraction protocols evaluated include the modified enzymatic extraction method (Method A), Chelex-100 extraction method (Method B), heat-induced retrieval in alkaline solution extraction method (Methods C and D) and one commercial FFPE DNA Extraction kit (Qiagen, Crawley, UK). For RNA extraction, 2 extraction protocols were evaluated including the enzymatic extraction method (Method 1), and Chelex-100 RNA extraction method (Method 2). Results show that the modified enzymatic extraction method (Method A) is an efficient DNA extraction protocol, while for RNA extraction, the enzymatic method (Method 1) and the Chelex-100 RNA extraction method (Method 2) are equally efficient RNA extraction protocols.

  6. RNA extraction from ten year old formalin-fixed paraffin-embedded breast cancer samples: a comparison of column purification and magnetic bead-based technologies

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    Zhang Haiyu


    Full Text Available Abstract Background The development of protocols for RNA extraction from paraffin-embedded samples facilitates gene expression studies on archival samples with known clinical outcome. Older samples are particularly valuable because they are associated with longer clinical follow up. RNA extracted from formalin-fixed paraffin-embedded (FFPE tissue is problematic due to chemical modifications and continued degradation over time. We compared quantity and quality of RNA extracted by four different protocols from 14 ten year old and 14 recently archived (three to ten months old FFPE breast cancer tissues. Using three spin column purification-based protocols and one magnetic bead-based protocol, total RNA was extracted in triplicate, generating 336 RNA extraction experiments. RNA fragment size was assayed by reverse transcription-polymerase chain reaction (RT-PCR for the housekeeping gene glucose-6-phosphate dehydrogenase (G6PD, testing primer sets designed to target RNA fragment sizes of 67 bp, 151 bp, and 242 bp. Results Biologically useful RNA (minimum RNA integrity number, RIN, 1.4 was extracted in at least one of three attempts of each protocol in 86–100% of older and 100% of recently archived ("months old" samples. Short RNA fragments up to 151 bp were assayable by RT-PCR for G6PD in all ten year old and months old tissues tested, but none of the ten year old and only 43% of months old samples showed amplification if the targeted fragment was 242 bp. Conclusion All protocols extracted RNA from ten year old FFPE samples with a minimum RIN of 1.4. Gene expression of G6PD could be measured in all samples, old and recent, using RT-PCR primers designed for RNA fragments up to 151 bp. RNA quality from ten year old FFPE samples was similar to that extracted from months old samples, but quantity and success rate were generally higher for the months old group. We preferred the magnetic bead-based protocol because of its speed and higher quantity of

  7. Validation of a PicoGreen-based DNA quantification integrated in an RNA extraction method for two-dimensional and three-dimensional cell cultures. (United States)

    Chen, Yantian; Sonnaert, Maarten; Roberts, Scott J; Luyten, Frank P; Schrooten, Jan


    DNA measurement and RNA extraction are two frequently used methods for cell characterization. In the conventional protocols, they require similar, but separate samples and in most cases, different pretreatments. The few combined protocols that exist still include time-consuming steps. Hence, to establish an efficient combined RNA extraction and DNA measurement protocol for two-dimensional (2D) and three-dimensional (3D) cell cultures, a PicoGreen-based DNA measurement was integrated in an existing RNA extraction protocol. It was validated by analysis of the influence of different lysis buffers, RLT, RA1, or Trizol, used for RNA extraction on the measured DNA concentration. The DNA cell yield was evaluated both in cell suspensions (2D) and on 3D cell-seeded scaffolds. Results showed that the different RNA lysis buffers caused a concentration-dependent perturbation of the PicoGreen signal. The measured DNA concentrations in 2D and 3D using RLT and RA1 buffer were comparable, also to the positive control. We, therefore, concluded that RNA extraction protocols using RA1 or RLT buffer allow the integration of a DNA quantification step without the buffer influencing the results. Hence, the combined DNA measurement and RNA extraction offer an alternative for DNA measurement techniques that is time and sample saving, for both 2D cell cultures and specific 3D constructs.

  8. Protocol: A simple phenol-based method for 96-well extraction of high quality RNA from Arabidopsis

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    Coustham Vincent


    Full Text Available Abstract Background Many experiments in modern plant molecular biology require the processing of large numbers of samples for a variety of applications from mutant screens to the analysis of natural variants. A severe bottleneck to many such analyses is the acquisition of good yields of high quality RNA suitable for use in sensitive downstream applications such as real time quantitative reverse-transcription-polymerase chain reaction (real time qRT-PCR. Although several commercial kits are available for high-throughput RNA extraction in 96-well format, only one non-kit method has been described in the literature using the commercial reagent TRIZOL. Results We describe an unusual phenomenon when using TRIZOL reagent with young Arabidopsis seedlings. This prompted us to develop a high-throughput RNA extraction protocol (HTP96 adapted from a well established phenol:chloroform-LiCl method (P:C-L that is cheap, reliable and requires no specialist equipment. With this protocol 192 high quality RNA samples can be prepared in 96-well format in three hours (less than 1 minute per sample with less than 1% loss of samples. We demonstrate that the RNA derived from this protocol is of high quality and suitable for use in real time qRT-PCR assays. Conclusion The development of the HTP96 protocol has vastly increased our sample throughput, allowing us to fully exploit the large sample capacity of modern real time qRT-PCR thermocyclers, now commonplace in many labs, and develop an effective high-throughput gene expression platform. We propose that the HTP96 protocol will significantly benefit any plant scientist with the task of obtaining hundreds of high quality RNA extractions.

  9. Comparison of three magnetic-bead-based RNA extraction methods for detection of cucumber green mottle mosaic virus by real-time RT-PCR. (United States)

    Zhao, Xiaoli; Zhou, Qi; Zhang, Lijie; Yan, Wenlong; Sun, Ning; Liang, Xinmiao; Deng, Congliang


    To determine the efficiency of RNA extraction methods based on magnetic beads, three different bead-based methods (one using silica-coated magnetic beads [SMNP], one using immunomagnetic beads conjugated to a specific antibody [IMB], and one using magnetic beads to nonspecifically adsorb virions [MNP]) were compared with the TRIzol method for the extraction of cucumber green mottle mosaic virus (CGMMV) RNA from cucumber leaves by real-time RT-PCR. The results indicated that the extraction efficiency of the SMNP method was 10 times higher than those of the IMB and MNP methods and 100 times higher than that of the TRIzol method. Therefore, the SMNP method could be considered for use in quarantine measures for the prevention and control of the disease caused by CGMMV.

  10. Extraction of total nucleic acid based on silica-coated magnetic particles for RT-qPCR detection of plant RNA virus/viroid. (United States)

    Sun, Ning; Deng, Congliang; Zhao, Xiaoli; Zhou, Qi; Ge, Guanglu; Liu, Yi; Yan, Wenlong; Xia, Qiang


    In this study, a nucleic acid extraction method based on silica-coated magnetic particles (SMPs) and RT-qPCR assay was developed to detect Arabis mosaic virus (ArMV), Lily symptomless virus (LSV), Hop stunt viroid (HSVd) and grape yellow speckle viroid 1 (GYSVd-1). The amplification sequences of RT-qPCR were reversely transcribed in vitro as RNA standard templates. The standard curves covered six or seven orders of magnitude with a detection limit of 100 copies per each assay. Extraction efficiency of the SMPs method was evaluated by recovering spiked ssRNAs from plant samples and compared to two commercial kits (TRIzol and RNeasy Plant mini kit). Results showed that the recovery rate of SMPs method was comparable to the commercial kits when spiked ssRNAs were extracted from lily leaves, whereas it was two or three times higher than commercial kits when spiked ssRNAs were extracted from grapevine leaves. SMPs method was also used to extract viral nucleic acid from15 ArMV-positive lily leaf samples and 15 LSV-positive lily leaf samples. SMPs method did not show statistically significant difference from other methods on detecting ArMV, but LSV. The SMPs method has the same level of virus load as the TRIzol, and its mean virus load of was 0.5log10 lower than the RNeasy Plant mini kit. Nucleic acid was extracted from 19 grapevine-leaf samples with SMPs and the two commercial kits and subsequently screened for HSVd and GYSVd-1 by RT-qPCR. Regardless of HSVd or GYSVd-1, SMPs method outperforms other methods on both positive rate and the viroid load. In conclusion, SMPs method was able to efficiently extract the nucleic acid of RNA viruses or viroids, especially grapevine viroids, from lily-leaf or grapevine-leaf samples for RT-qPCR detection.

  11. PRI-Modeler: extracting RNA structural elements from PDB files of protein-RNA complexes. (United States)

    Han, Kyungsook; Nepal, Chirag


    A complete understanding of protein and RNA structures and their interactions is important for determining the binding sites in protein-RNA complexes. Computational approaches exist for identifying secondary structural elements in proteins from atomic coordinates. However, similar methods have not been developed for RNA, due in part to the very limited structural data so far available. We have developed a set of algorithms for extracting and visualizing secondary and tertiary structures of RNA and for analyzing protein-RNA complexes. These algorithms have been implemented in a web-based program called PRI-Modeler (protein-RNA interaction modeler). Given one or more protein data bank files of protein-RNA complexes, PRI-Modeler analyzes the conformation of the RNA, calculates the hydrogen bond (H bond) and van der Waals interactions between amino acids and nucleotides, extracts secondary and tertiary RNA structure elements, and identifies the patterns of interactions between the proteins and RNAs. This paper presents PRI-Modeler and its application to the hydrogen bond and van der Waals interactions in the most representative set of protein-RNA complexes. The analysis reveals several interesting interaction patterns at various levels. The information provided by PRI-Modeler should prove useful for determining the binding sites in protein-RNA complexes. PRI-Modeler is accessible at, and supplementary materials are available in the analysis results section at




    ABSTRACT Functional genomic analyses require intact RNA; however, Passiflora edulis leaves are rich in secondary metabolites that interfere with RNA extraction primarily by promoting oxidative processes and by precipitating with nucleic acids. This study aimed to analyse three RNA extraction methods, Concert™ Plant RNA Reagent (Invitrogen, Carlsbad, CA, USA), TRIzol® Reagent (Invitrogen) and TRIzol® Reagent (Invitrogen)/ice -commercial products specifically designed to extract RNA, and...

  13. RNA Contaminates Glycosaminoglycans Extracted from Cells and Tissues (United States)

    de Graaf, Mark J. J.; Berden, Jo H. M.; Rabelink, Ton J.; Smit, Cornelis H.


    Glycosaminoglycans (GAGs) are linear negatively charged polysaccharides and important components of extracellular matrices and cell surface glycan layers such as the endothelial glycocalyx. The GAG family includes sulfated heparin, heparan sulfate (HS), dermatan sulfate (DS), chondroitin sulfate (CS), keratan sulfate, and non-sulfated hyaluronan. Because relative expression of GAGs is dependent on cell-type and niche, isolating GAGs from cell cultures and tissues may provide insight into cell- and tissue-specific GAG structure and functions. In our objective to obtain structural information about the GAGs expressed on a specialized mouse glomerular endothelial cell culture (mGEnC-1) we adapted a recently published GAG isolation protocol, based on cell lysis, proteinase K and DNase I digestion. Analysis of the GAGs contributing to the mGEnC-1 glycocalyx indicated a large HS and a minor CS content on barium acetate gel. However, isolated GAGs appeared resistant to enzymatic digestion by heparinases. We found that these GAG extracts were heavily contaminated with RNA, which co-migrated with HS in barium acetate gel electrophoresis and interfered with 1,9-dimethylmethylene blue (DMMB) assays, resulting in an overestimation of GAG yields. We hypothesized that RNA may be contaminating GAG extracts from other cell cultures and possibly tissue, and therefore investigated potential RNA contaminations in GAG extracts from two additional cell lines, human umbilical vein endothelial cells and retinal pigmental epithelial cells, and mouse kidney, liver, spleen and heart tissue. GAG extracts from all examined cell lines and tissues contained varying amounts of contaminating RNA, which interfered with GAG quantification using DMMB assays and characterization of GAGs by barium acetate gel electrophoresis. We therefore recommend routinely evaluating the RNA content of GAG extracts and propose a robust protocol for GAG isolation that includes an RNA digestion step. PMID:27898729

  14. Rapid methods to extract DNA and RNA from Cryptococcus neoformans. (United States)

    Bolano, A; Stinchi, S; Preziosi, R; Bistoni, F; Allegrucci, M; Baldelli, F; Martini, A; Cardinali, G


    Extraction of nucleic acids from the pathogenic yeast Cryptococcus neoformans is normally hampered by a thick and resistant capsule, accounting for at least 70% of the whole cellular volume. This paper presents procedures based on mechanical cell breakage to extract DNA and RNA from C. neoformans and other capsulated species. The proposed system for DNA extraction involves capsule relaxation by means of a short urea treatment and bead beating. These two steps allow a consistent extraction even from strains resistant to other procedures. Yield and quality of DNA obtained with the proposed method were higher than those obtained with two earlier described methods. This protocol can be extended to every yeast species and particularly to those difficult to handle for the presence of a capsule. RNA purification is accomplished using an original lysing matrix and the FastPrep System (Bio101) after a preliminary bead beating treatment. Yields range around 1 mg RNA from 15 ml overnight culture (10(9) cells), RNA appears undegraded, making it suitable for molecular manipulations.

  15. Assessing an improved protocol for plasma microRNA extraction.

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    Inés Moret

    Full Text Available The first step in biomarkers discovery is to identify the best protocols for their purification and analysis. This issue is critical when considering peripheral blood samples (plasma and serum that are clinically interesting but meet several methodological problems, mainly complexity and low biomarker concentration. Analysis of small molecules, such as circulating microRNAs, should overcome these disadvantages. The present study describes an optimal RNA extraction method of microRNAs from human plasma samples. Different reagents and commercially available kits have been analyzed, identifying also the best pre-analytical conditions for plasma isolation. Between all of them, the column-based approaches were shown to be the most effective. In this context, miRNeasy Serum/Plasma Kit (from Qiagen rendered more concentrated RNA, that was better suited for microarrays studies and did not require extra purification steps for sample concentration and purification than phenol based extraction methods. We also present evidences that the addition of low doses of an RNA carrier before starting the extraction process improves microRNA purification while an already published carrier dose can result in significant bias over microRNA profiles. Quality controls for best protocol selection were developed by spectrophotometry measurement of contaminants and microfluidics electrophoresis (Agilent 2100 Bioanalyzer for RNA integrity. Selected donor and patient plasma samples and matched biopsies were tested by Affymetrix microarray technology to compare differentially expressed microRNAs. In summary, this study defines an optimized protocol for microRNA purification from human blood samples, increasing the performance of assays and shedding light over the best way to discover and use these biomarkers in clinical practice.

  16. Comparison of DNA and RNA extraction methods for mummified tissues. (United States)

    Konomi, Nami; Lebwohl, Eve; Zhang, David


    Nucleic acids extracted from mummified tissues are valuable materials for the study of ancient human beings. Significant difficulty in extracting nucleic acids from mummified tissues has been reported due to chemical modification and degradation. The goal of this study was to determine a method that is more efficient for DNA and RNA extraction from mummified tissues. Twelve mummy specimens were analyzed with 9 different nucleic acid extraction methods, including guanidium thiocyanate (GTC) and proteinase K/detergent based methods prepared in our laboratory or purchased. Glyceraldehyde 3-phosphate dehydrogenase DNA and beta-actin RNA were used as markers for the presence of adequate DNA and RNA, respectively, for PCR and RT-PCR amplification. Our results show that 5 M GTC is more efficient of releasing nucleic acids from mummified tissue than proteinase K/detergent, and phenol/chloroform extraction with an additional chloroform step is more efficient than phenol/chloroform along. We were able to isolate DNAs from all 12 specimens and RNAs from 8 of 12 specimens, and the nucleic acids were sufficient for PCR and RT-PCR analysis. We further tested hepatitis viruses including hepatitis B virus, hepatitis C virus, hepatitis G virus, and TT virus DNA, and fail to detect these viruses in all 12 specimens.

  17. Protocol: A simple phenol-based method for 96-well extraction of high quality RNA from Arabidopsis



    Abstract Background Many experiments in modern plant molecular biology require the processing of large numbers of samples for a variety of applications from mutant screens to the analysis of natural variants. A severe bottleneck to many such analyses is the acquisition of good yields of high quality RNA suitable for use in sensitive downstream applications such as real time quantitative reverse-transcription-polymerase chain reaction (real time qRT-PCR). Although several commercial kits are a...


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    Full Text Available ABSTRACT Functional genomic analyses require intact RNA; however, Passiflora edulis leaves are rich in secondary metabolites that interfere with RNA extraction primarily by promoting oxidative processes and by precipitating with nucleic acids. This study aimed to analyse three RNA extraction methods, Concert™ Plant RNA Reagent (Invitrogen, Carlsbad, CA, USA, TRIzol® Reagent (Invitrogen and TRIzol® Reagent (Invitrogen/ice -commercial products specifically designed to extract RNA, and to determine which method is the most effective for extracting RNA from the leaves of passion fruit plants. In contrast to the RNA extracted using the other 2 methods, the RNA extracted using TRIzol® Reagent (Invitrogen did not have acceptable A260/A280 and A260/A230 ratios and did not have ideal concentrations. Agarose gel electrophoresis showed a strong DNA band for all of the Concert™ method extractions but not for the TRIzol® and TRIzol®/ice methods. The TRIzol® method resulted in smears during electrophoresis. Due to its low levels of DNA contamination, ideal A260/A280 and A260/A230 ratios and superior sample integrity, RNA from the TRIzol®/ice method was used for reverse transcription-polymerase chain reaction (RT-PCR, and the resulting amplicons were highly similar. We conclude that TRIzol®/ice is the preferred method for RNA extraction for P. edulis leaves.

  19. Optimization of RNA Extraction from Rat Pancreatic Tissue

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    Sanaz Dastgheib


    Full Text Available Background: Optimized RNA extraction from tissues and cell lines consists of four main stages regardless of the method of extraction: 1 homogenizing, 2 effective denaturation of proteins from RNA, 3 inactivation of ribonuclease, and 4 removal of any DNA, protein, and carbohydrate contamination. Isolation of undamaged intact RNA is challenging when the related tissue contains high levels of RNase. Various technical difficulties occur during extraction of RNA from pancreatic tissue due to spontaneous autolysis. Since standard routine protocols yield unacceptable results in pancrease, we have designed a simple method for RNA extraction by comparing different protocols. Methods: We obtained 20-30 mg pancreatic tissues in less than 2 min from 30 rats. Several methods were performed to extract RNA from pancreatic tissue and evaluate its integrity. All methods were performed three times to obtain reproducible results. Results: Immersing pancreatic tissue in RNA-later for 24 h at -80ºC yielded high quality RNA by using the TriPure reagent which was comparable to the commercial RNeasy Micro Kit. The quality of RNA was evaluated by spectrophotometer, electrophoresis and RT-PCR. We separated intact 28S and 18S ribosomal RNA (rRNA when our procedure was compared with the RNeasy Micro Kit. Finally, full length of the actin gene was amplified by RT-PCR. Conclusion: We designed a simple, fast, cost-effective method for complete RNA extraction from the least amount of quantitatively intact pancreatic tissue

  20. Immunotherapy of intracranial G422 glioblastoma with dendritic cells pulsed with tumor extract or RNA

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    张哲; 汤灵玲; 詹仁雅; 童鹰; 姚航平; 杜理安


    Objective: To investigate the anti-tumor efficacy of dendritic cell (DC)-based vaccines pulsed with tumor extracts or RNA in a mouse model of intracranial G422 glioblastoma. Methods: Bone marrow-derived DCs were pulsed ex vivo with tumor extracts or RNA. Ninety female mice harboring 4-day-old intracranial G422 glioblastomas and 126 normal mice were treated with three spaced one week apart subcutaneous injections either with PBS, unpulsed DCs, G422 tumor extracts, RNA, DCs pulsed with G422 tumor extracts (DC/extract) or with RNA (DC/RNA). Seven days after the third immunization of normal mice, the spleens of 36 of them were harvested for cytotoxic T lyphocyte (CTL) assays and the others were challenged in the brain with G422 tumor cells. All the treated mice were followed for survival. Some mice brains were removed and examined pathologically when they died. Results: Immunization using DC/extract or DC/RNA significantly induced G422-specific CTL responses compared with control groups (P<0.01). Vaccination with DC/extract or DC/RNA, either prior to G422 tumor challenge or in tumor-harboring mice, significantly prolonged survival compared with other control groups (P<0.01). Conclusion: DCs pulsed with tumor extracts or RNA derived from autologous tumors has potential antitumor effects via activation of cell-mediated immunity. Our results suggest a useful therapeutic strategy against gliomas.

  1. Immunotherapy of intracranial G422 glioblastoma with dendritic cells pulsed with tumor extract or RNA

    Institute of Scientific and Technical Information of China (English)

    张哲; 汤灵玲; 詹仁雅; 童鹰; 姚航平; 杜理安


    Objective: To investigate the anti-tumor efficacy of dendritic cell (DC)-based vaccines pulsed with tumor extracts or RNA in a mouse model of intracranial G422 glioblastoma. Methods: Bone marrow-derived DCs were pulsed ex vivo with tumor extracts or RNA. Ninety female mice harboring 4-day-old intracranial G422 glioblastomas and 126 normal mice were treated with three spaced one week apart subcutaneous injections either with PBS, unpulsed DCs, G422 tumor extracts, RNA, DCs pulsed with G422 tumor extracts (DC/extract) or with RNA (DC/RNA). Seven days after the third immunization of normal mice, the spleens of 36 of them were harvested for cytotoxic T lyphocyte (CTL) assays and the others were challenged in the brain with G422 tumor cells. All the treated mice were followed for survival. Some mice brains were removed and examined pathologically when they died. Results: Immunization using DC/extract or DC/RNA significantly induced G422-specific CTL responses compared with control groups (P<0.01). Vaccination with DC/extract or DC/RNA, either prior to G422 tumor challenge or in tumor-harboring mice, significantly prolonged survival compared with other control groups (P<0.01). Conclusion: DCs pulsed with tumor extracts or RNA derived from autologous tumors has potential antitumor effects via activation of cell-mediated immunity. Our results suggest a useful therapeutic strategy against gliomas.

  2. Integrated DNA and RNA extraction using magnetic beads from viral pathogens causing acute respiratory infections (United States)

    He, Hui; Li, Rongqun; Chen, Yi; Pan, Ping; Tong, Wenjuan; Dong, Xueyan; Chen, Yueming; Yu, Daojun


    Current extraction methods often extract DNA and RNA separately, and few methods are capable of co-extracting DNA and RNA from sputum. We established a nucleic acid co-extraction method from sputum based on magnetic beads and optimized the method by evaluating influencing factors, such as the guanidinium thiocyanate (GTC) and dithiothreitol (DTT) concentrations, magnetic bead amount, incubation temperature, lysis buffer pH and RNA carrier type. The feasibility of the simultaneous nucleic acid co-extraction method was evaluated by amplifying DNA and RNA viruses from a single clinical specimen with a multiplex RT-qPCR method. Both DNA and RNA were most efficiently extracted when the GTC and DTT concentrations were 2.0 M and 80 mM, respectively, 20 μl magnetic beads were added, the incubation temperature was 80 °C, the pH was 8 or 9, and RNA carrier A was used. Therefore, we established a simple method to extract nucleic acids from two important respiratory viruses compared with other commercial kits. This magnetic beads-based co-extraction method for sputum followed by a multiplex RT-qPCR can rapidly and precisely detect DNA and RNA viruses from a single clinical specimen and has many advantages, such as decreased time, low cost, and a lack of harmful chemicals. PMID:28332631

  3. A non-phenol-chloroform extraction of double-stranded RNA from plant and fungal tissues. (United States)

    Balijja, Alitukiriza; Kvarnheden, Anders; Turchetti, Tullio


    Double-stranded RNA (dsRNA) molecules of viruses are found in nature at a very high frequency. Their detection in plants and fungi has been carried out with difficulty due to the complicated dsRNA extraction techniques used commonly which includes phenol-chloroform extractions. In this study, an extraction method for isolation of dsRNA is described that is free of phenol and chloroform. A lysis buffer, containing beta-mercaptoethanol and polyvinylpolypyrrolidone (PVPP-40), was added to homogenised tissues and the subsequent supernatant was filtered through a cellulose CF-11 mini-column. DsRNA molecules were separated based on the differing affinity of nucleic acids for the cellulose CF-11 resin in 20% ethanol buffer. This easy, rapid and cheap technique has been successfully tested on fungi and plants containing different dsRNA virus molecules, indicating the possibility of a wide use of the method.

  4. An efficient RNA extraction method for estimating gut microbial diversity by polymerase chain reaction. (United States)

    Kang, Seungha; Denman, Stuart E; Morrison, Mark; Yu, Zhongtang; McSweeney, Chris S


    An extraction method was developed to recover high-quality RNA from rumen digesta and mouse feces for phylogenetic analysis of metabolically active members of the gut microbial community. Four extraction methods were tested on different amounts of the same samples and compared for efficiency of recovery and purity of RNA. Trizol extraction after bead beating produced a higher quantity and quality of RNA than a similar method using phenol/chloroform. Dissociation solution produced a 1.5- to 2-fold increase in RNA recovery compared with phosphate-buffered saline during the dissociation of microorganisms from rumen digesta or fecal particles. The identity of metabolically active bacteria in the samples was analyzed by sequencing 87 amplicons produced using bacteria-specific 16S rDNA primers, with cDNA synthesized from the extracted RNA as the template. Amplicons representing the major phyla encountered in the rumen (Firmicutes, 43.7%; Proteobacteria, 28.7%; Bacteroidetes, 25.3%; Spirochea, 1.1%, and Synergistes, 1.1%) were recovered, showing that development of the RNA extraction method enables RNA-based analysis of metabolically active bacterial groups from the rumen and other environments. Interestingly, in rumen samples, about 30% of the sequenced random 16S rRNA amplicons were related to the Proteobacteria, providing the first evidence that this group may have greater importance in rumen metabolism than previously attributed by DNA-based analysis.

  5. Comparison of RNA extraction methods from biofilm samples of Staphylococcus epidermidis

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    França Angela


    Full Text Available Abstract Background Microbial biofilms are communities of bacteria adhered to a surface and surrounded by an extracellular polymeric matrix. Biofilms have been associated with increased antibiotic resistance and tolerance to the immune system. Staphylococcus epidermidis is the major bacterial species found in biofilm-related infections on indwelling medical devices. Obtaining high quality mRNA from biofilms is crucial to validate the transcriptional measurements associated with the switching to the biofilm mode of growth. Therefore, we selected three commercially available RNA extraction kits with distinct characteristics, including those using silica membrane or organic extraction methods, and enzymatic or mechanical cell lysis, and evaluated the RNA quality obtained from two distinct S. epidermidis bacterial biofilms. Results RNA extracted using the different kits was evaluated for quantity, purity, integrity, and functionally. All kits were able to extract intact and functional total RNA from the biofilms generated from each S. epidermidis strain. The results demonstrated that the kit based on mechanical lysis and organic extraction (FastRNA® Pro Blue was the only one that was able to isolate pure and large quantities of RNA. Normalized expression of the icaA virulence gene showed that RNA extracted with PureLink™ had a significant lower concentration of icaA mRNA transcripts than the other kits tested. Conclusions When working with complex samples, such as biofilms, that contain a high content extracellular polysaccharide and proteins, special care should be taken when selecting the appropriate RNA extraction system, in order to obtain accurate, reproducible, and biologically significant results. Among the RNA extraction kits tested, FastRNA® Pro Blue was the best option for both S. epidermidis biofilms used.

  6. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development (United States)

    The bacterial composition of chlorinated drinking water was analyzed using 16S rRNA gene clone libraries derived from DNA extracts of 12 samples and compared to clone libraries previously generated using RNA extracts from the same samples. Phylogenetic analysis of 761 DNA-based ...

  7. Automated extraction protocol for quantification of SARS-Coronavirus RNA in serum: an evaluation study

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    Lui Wing-bong


    Full Text Available Abstract Background We have previously developed a test for the diagnosis and prognostic assessment of the severe acute respiratory syndrome (SARS based on the detection of the SARS-coronavirus RNA in serum by real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR. In this study, we evaluated the feasibility of automating the serum RNA extraction procedure in order to increase the throughput of the assay. Methods An automated nucleic acid extraction platform using the MagNA Pure LC instrument (Roche Diagnostics was evaluated. We developed a modified protocol in compliance with the recommended biosafety guidelines from the World Health Organization based on the use of the MagNA Pure total nucleic acid large volume isolation kit for the extraction of SARS-coronavirus RNA. The modified protocol was compared with a column-based extraction kit (QIAamp viral RNA mini kit, Qiagen for quantitative performance, analytical sensitivity and precision. Results The newly developed automated protocol was shown to be free from carry-over contamination and have comparable performance with other standard protocols and kits designed for the MagNA Pure LC instrument. However, the automated method was found to be less sensitive, less precise and led to consistently lower serum SARS-coronavirus concentrations when compared with the column-based extraction method. Conclusion As the diagnostic efficiency and prognostic value of the serum SARS-CoV RNA RT-PCR test is critically associated with the analytical sensitivity and quantitative performance contributed both by the RNA extraction and RT-PCR components of the test, we recommend the use of the column-based manual RNA extraction method.

  8. Automated serial extraction of DNA and RNA from biobanked tissue specimens


    Mathot, Lucy; Wallin, Monica; Sjöblom, Tobias


    Background: With increasing biobanking of biological samples, methods for large scale extraction of nucleic acids are in demand. The lack of such techniques designed for extraction from tissues results in a bottleneck in downstream genetic analyses, particularly in the field of cancer research. We have developed an automated procedure for tissue homogenization and extraction of DNA and RNA into separate fractions from the same frozen tissue specimen. A purpose developed magnetic bead based te...

  9. Ultrasensitive detection of mRNA extracted from cancerous cells achieved by DNA rotaxane-based cross-rolling circle amplification. (United States)

    Bi, Sai; Cui, Yangyang; Li, Li


    An ultrasensitive and highly selective method for polymerase chain reaction-free (PCR-free) messenger RNA (mRNA) expression profiling is developed through a novel cross-rolling circle amplification (C-RCA) process based on DNA-rotaxane nanostructures. Two species of DNA pseudorotaxane (DPR) superstructures (DPR-I and DPR-II) are assembled by threading a linear DNA rod through a double-stranded DNA (dsDNA) ring containing two single-stranded gaps. In this assay, cDNA that is specific for β-actin (ACTB) mRNA is taken as a model analyte. Upon the introduction of the target cDNA, the cDNA and the biotin-modified primer are hybridized to the single-stranded regions of the DNA rod and the gap-ring, respectively. As a result, the DPR-I dethreads into free DNA macrocycle and a dumbbell-shaped DNA nanostructure. In the presence of DNA polymerase/dNTPs, two release-DNA on the DPR-I are replaced by polymerase with strand-displacement activity, which can act as the input of the DPR-II to trigger the dethreading of DPR-II and the RCA reaction, releasing another two specified release-DNA strands those in turn serve as the "mimic cDNA" for DPR-I. The C-RCA reaction then proceeds autonomously. To overcome the high background induced by hemin itself, the biotinylated rolling circle products are captured by streptavidin-coated MNPs, achieving a detection limit as low as 0.1 zmol cDNA. The assay also exhibits an excellent selectivity due to its unique DNA nanostructure fabricated through base pairing hybridization. The ACTB mRNA expression in mammary cancer cells (MCF-7) is successfully detected.

  10. Extraction of HCV-RNA from Plasma Samples: Development towards Semiautomation


    Imran Amin; Tania Jabbar; Fawad Niazi; Muhammad Saeed Akhtar


    A semiautomated extraction protocol of HCV-RNA using Favorgen RNA extraction kit has been developed. The kit provided protocol was modified by replacing manual spin steps with vacuum filtration. The assay performance was evaluated by real-time qPCR based on Taqman technology. Assay linearity was confirmed with the serial dilutions of RTA (Turkey) containing 1 × (106, 105, 104, and 103) IU mL−1. Comparison of test results obtained by two extraction methods showed a good correlation (r = 0.95, ...

  11. Comparison of RNA extraction methods in Thai aromatic coconut water

    Directory of Open Access Journals (Sweden)

    Nopporn Jaroonchon


    Full Text Available Many researches have reported that nucleic acid in coconut water is in free form and at very low yields which makes it difficult to process in molecular studies. Our research attempted to compare two extraction methods to obtain a higher yield of total RNA in aromatic coconut water and monitor its change at various fruit stages. The first method used ethanol and sodium acetate as reagents; the second method used lithium chloride. We found that extraction using only lithium chloride gave a higher total RNA yield than the method using ethanol to precipitate nucleic acid. In addition, the total RNA from both methods could be used in amplification of betaine aldehyde dehydrogenase2 (Badh2 genes, which is involved in coconut aroma biosynthesis, and could be used to perform further study as we expected. From the molecular study, the nucleic acid found in coconut water increased with fruit age.

  12. Urinary extracellular vesicles for RNA extraction: optimization of a protocol devoid of prokaryote contamination

    Directory of Open Access Journals (Sweden)

    Dorota Tataruch-Weinert


    Full Text Available Background: Urinary extracellular vesicles (UEVs represent an ideal platform for biomarker discovery. They carry different types of RNA species, and reported profile discrepancies related to the presence/absence of 18s and 28s rRNA remain controversial. Moreover, sufficient urinary RNA yields and respective quality RNA profiles are still to be fully established. Methods: UEVs were enriched by hydrostatic filtration dialysis, and RNA content was extracted using 7 different commercially available techniques. RNA quantity was assessed using spectrophotometry and fluorometry, whilst RNA quality was determined by capillary electrophoresis. Results: The presence of prokaryotic transcriptome was stressed when cellular RNA, as a control, was spiked into the UEVs samples before RNA extraction. The presence of bacteria in hydrostatic filtration dialysis above 1,000 kDa molecular weight cut-off and in crude urine was confirmed with growth media plates. The efficiency in removing urinary bacteria was evaluated by differential centrifugation, filtration (0.22 µm filters and chemical pretreatment (water purification tablet. For volumes of urine >200 ml, the chemical treatment provides ease of handling without affecting vesicle integrity, protein and RNA profiles. This protocol was selected to enrich RNA with 7 methods, and its respective quality and quantity were assessed. The results were given as follows: (a Fluorometry gave more repeatability and reproducibility than spectrophotometry to assess the RNA yields, (b UEVs were enriched with small RNA, (c Ribosomal RNA peaks were not observed for any RNA extraction method used and (d RNA yield was higher for column-based method designed for urinary exosome, whilst the highest relative microRNA presence was obtained using TRIzol method. Conclusion: Our results show that the presence of bacteria can lead to misidentification in the electrophoresis peaks. Fluorometry is more reliable than spectrophotometry. RNA

  13. Molecular Techniques for Dicistrovirus Detection without RNA Extraction or Purification

    Directory of Open Access Journals (Sweden)

    Jailson F. B. Querido


    Full Text Available Dicistroviridae is a new family of small, nonenveloped, and +ssRNA viruses pathogenic to both beneficial arthropods and insect pests as well. Triatoma virus (TrV, a dicistrovirus, is a pathogen of Triatoma infestans (Hemiptera: Reduviidae, one of the main vectors of Chagas disease. In this work, we report a single-step method to identify TrV, a dicistrovirus, isolated from fecal samples of triatomines. The identification method proved to be quite sensitive, even without the extraction and purification of RNA virus.

  14. RNA-RNA interaction prediction based on multiple sequence alignments

    CERN Document Server

    Li, Andrew X; Qin, Jing; Reidys, Christian M


    Recently, $O(N^6)$ time and $O(N^4)$ space dynamic programming algorithms have become available that compute the partition function of RNA-RNA interaction complexes for pairs of RNA sequences. These algorithms and the biological requirement of more reliable interactions motivate to utilize the additional information contained in multiple sequence alignments and to generalize the above framework to the partition function and base pairing probabilities for multiple sequence alignments.

  15. Extraction of total RNA in the developing chicken forebrain

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    Sayed Rasoul Zaker


    Full Text Available Background: Gene expression of Gama-Aminobutyric acid (GABA A receptor subunits may change during development. Procedures in molecular biology are required to understand the gene expression profile GABA A R in chicken. The outcome of the results depends on good-quality high-molecular-weight RNA. Several procedures can be used to isolate RNA from the brain of chicken; however, most of them are time-consuming and require disruption of cells or freeze and thaw in the presence of RNase inhibitors. The aim of this experiment was isolation of RNA from chicken embryonic brain tissues using appropriate RNA extraction kit. Materials and Methods: Fertilized eggs from Ross breed (Gallus gallus were incubated at 38°C and 60% relative humidity in a forced-draft incubator and were turned every 3 h. After 3, 7, 14 and 20 days of incubation, eggs were cooled on ice to induce deep anesthesia. Then whole brains were dissected out. As brains could not be excised in a reproducible way from earlier embryos (embryonic days 4 and 6, whole heads were collected. Chicken embryos between day 7 to 20 and 1 day after birth were decapitated, and their brains removed. Samples were immediately inserted into lysis buffer and stored at −70°C. Total RNA was isolated and a contaminating genomic deoxyribonucleic acid (DNA was digested. RNA quality was checked using gel electrophoresis. Results: We obtained 52 mg/ml to 745 mg/ml with A260/280 1.7-2.2. Only high-quality RNA, with no signs of degradation, was used for further experiments. Conclusion: In conclusion, protocol was found to be suitable for the isolation of total RNA from embryonic chicken cells.

  16. Efficient and scalable serial extraction of DNA and RNA from frozen tissue samples. (United States)

    Mathot, Lucy; Lindman, Monica; Sjöblom, Tobias


    Advances in cancer genomics have created a demand for scalable sample processing. We here present a process for serial extraction of nucleic acids from the same frozen tissue sample based on magnetic silica particles. The process is automation friendly with high recoveries of pure DNA and RNA suitable for analysis.

  17. Comparison of commercial RNA extraction kits for preparation of DNA-free total RNA from Salmonella cells

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    Gonzalez-Escalona Narjol


    Full Text Available Abstract Background The isolation of DNA-free RNA is a crucial step in the reverse transcription PCR (RT-PCR. Every RNA extraction procedure results in RNA samples contaminated with genomic DNA, which can cause false-positive outcomes in highly sensitive applications, including a recently developed quantitative real-time PCR (RT-qPCR assay that targets invA mRNA for the detection of live Salmonella cells. The assay of this specific mRNA can be used to indicate the presence of live, as opposed to dead, cells of Salmonella enterica in a food matrix. Findings We evaluated the ability of five RNA extraction kits to produce RNA preparations from exponentially growing Salmonella cells. The acceptability of the preparations for use in downstream applications such as RT-qPCR was judged in terms of the total amount of RNA recovered, the integrity of the RNA molecules, and minimal content of DNA. The five kits produced RNA preparations that differed markedly in yield, integrity of the Salmonella RNA and the amount of contaminant DNA. The greatest RNA recovery was achieved with the MasterPure kit; however, the preparation contained high levels of genomic DNA. The UltraClean extraction kit gave a low level of RNA recovery with a poor level of integrity. The RNeasy Mini, RiboPure and PureLink extraction kits produced high-quality, DNA-free RNA suitable for Salmonella detection by RT-qPCR. Conclusions We showed that the RNeasy Mini and PureLink RNA extraction kits were the most suitable for the detection of Salmonella invA mRNA by RT-qPCR. The use of these two kits will greatly reduce the frequency of false-positive results and might allow fast RT-qPCR determination of invA mRNA produced by viable Salmonella in food samples.

  18. High quality RNA extraction from Maqui berry for its application in next-generation sequencing. (United States)

    Sánchez, Carolina; Villacreses, Javier; Blanc, Noelle; Espinoza, Loreto; Martinez, Camila; Pastor, Gabriela; Manque, Patricio; Undurraga, Soledad F; Polanco, Victor


    Maqui berry (Aristotelia chilensis) is a native Chilean species that produces berries that are exceptionally rich in anthocyanins and natural antioxidants. These natural compounds provide an array of health benefits for humans, making them very desirable in a fruit. At the same time, these substances also interfere with nucleic acid preparations, making RNA extraction from Maqui berry a major challenge. Our group established a method for RNA extraction of Maqui berry with a high quality RNA (good purity, good integrity and higher yield). This procedure is based on the adapted CTAB method using high concentrations of PVP (4 %) and β-mercaptoethanol (4 %) and spermidine in the extraction buffer. These reagents help to remove contaminants such as polysaccharides, proteins, phenols and also prevent the oxidation of phenolic compounds. The high quality of RNA isolated through this method allowed its uses with success in molecular applications for this endemic Chilean fruit, such as differential expression analysis of RNA-Seq data using next generation sequencing (NGS). Furthermore, we consider that our method could potentially be used for other plant species with extremely high levels of antioxidants and anthocyanins.

  19. ncRNA-class Web Tool: Non-coding RNA feature extraction and pre-miRNA classification web tool

    KAUST Repository

    Kleftogiannis, Dimitrios A.


    Until recently, it was commonly accepted that most genetic information is transacted by proteins. Recent evidence suggests that the majority of the genomes of mammals and other complex organisms are in fact transcribed into non-coding RNAs (ncRNAs), many of which are alternatively spliced and/or processed into smaller products. Non coding RNA genes analysis requires the calculation of several sequential, thermodynamical and structural features. Many independent tools have already been developed for the efficient calculation of such features but to the best of our knowledge there does not exist any integrative approach for this task. The most significant amount of existing work is related to the miRNA class of non-coding RNAs. MicroRNAs (miRNAs) are small non-coding RNAs that play a significant role in gene regulation and their prediction is a challenging bioinformatics problem. Non-coding RNA feature extraction and pre-miRNA classification Web Tool (ncRNA-class Web Tool) is a publicly available web tool ( ) which provides a user friendly and efficient environment for the effective calculation of a set of 58 sequential, thermodynamical and structural features of non-coding RNAs, plus a tool for the accurate prediction of miRNAs. © 2012 IFIP International Federation for Information Processing.

  20. Extracting microRNA-gene relations from biomedical literature using distant supervision (United States)

    Clarke, Luka A.; Couto, Francisco M.


    Many biomedical relation extraction approaches are based on supervised machine learning, requiring an annotated corpus. Distant supervision aims at training a classifier by combining a knowledge base with a corpus, reducing the amount of manual effort necessary. This is particularly useful for biomedicine because many databases and ontologies have been made available for many biological processes, while the availability of annotated corpora is still limited. We studied the extraction of microRNA-gene relations from text. MicroRNA regulation is an important biological process due to its close association with human diseases. The proposed method, IBRel, is based on distantly supervised multi-instance learning. We evaluated IBRel on three datasets, and the results were compared with a co-occurrence approach as well as a supervised machine learning algorithm. While supervised learning outperformed on two of those datasets, IBRel obtained an F-score 28.3 percentage points higher on the dataset for which there was no training set developed specifically. To demonstrate the applicability of IBRel, we used it to extract 27 miRNA-gene relations from recently published papers about cystic fibrosis. Our results demonstrate that our method can be successfully used to extract relations from literature about a biological process without an annotated corpus. The source code and data used in this study are available at PMID:28263989

  1. Automated Device for Asynchronous Extraction of RNA, DNA, or Protein Biomarkers from Surrogate Patient Samples. (United States)

    Bitting, Anna L; Bordelon, Hali; Baglia, Mark L; Davis, Keersten M; Creecy, Amy E; Short, Philip A; Albert, Laura E; Karhade, Aditya V; Wright, David W; Haselton, Frederick R; Adams, Nicholas M


    Many biomarker-based diagnostic methods are inhibited by nontarget molecules in patient samples, necessitating biomarker extraction before detection. We have developed a simple device that purifies RNA, DNA, or protein biomarkers from complex biological samples without robotics or fluid pumping. The device design is based on functionalized magnetic beads, which capture biomarkers and remove background biomolecules by magnetically transferring the beads through processing solutions arrayed within small-diameter tubing. The process was automated by wrapping the tubing around a disc-like cassette and rotating it past a magnet using a programmable motor. This device recovered biomarkers at ~80% of the operator-dependent extraction method published previously. The device was validated by extracting biomarkers from a panel of surrogate patient samples containing clinically relevant concentrations of (1) influenza A RNA in nasal swabs, (2) Escherichia coli DNA in urine, (3) Mycobacterium tuberculosis DNA in sputum, and (4) Plasmodium falciparum protein and DNA in blood. The device successfully extracted each biomarker type from samples representing low levels of clinically relevant infectivity (i.e., 7.3 copies/µL of influenza A RNA, 405 copies/µL of E. coli DNA, 0.22 copies/µL of TB DNA, 167 copies/µL of malaria parasite DNA, and 2.7 pM of malaria parasite protein).

  2. Comparison of methods for miRNA extraction from plasma and quantitative recovery of RNA from plasma and cerebrospinal fluid

    Directory of Open Access Journals (Sweden)

    Melissa A McAlexander


    Full Text Available Interest in extracellular RNA has intensified as evidence accumulates that these molecules may be useful as indicators of a wide variety of biological conditions. To establish specific extracellular RNA molecules as clinically relevant biomarkers, reproducible recovery from biological samples and reliable measurements of the isolated RNA are paramount. Towards these ends, careful and rigorous comparisons of technical procedures are needed at all steps from sample handling to RNA isolation to RNA measurement protocols. In the investigations described in this methods paper, RT-qPCR was used to examine the apparent recovery of specific endogenous miRNAs and a spiked-in synthetic RNA from blood plasma samples. RNA was isolated using several widely used RNA isolation kits, with or without the addition of glycogen as a carrier. Kits examined included total RNA isolation systems that have been commercially available for several years and commonly adapted for extraction of biofluid RNA, as well as more recently introduced biofluids-specific RNA methods. Our conclusions include the following: some RNA isolation methods appear to be superior to others for the recovery of RNA from biological fluids; addition of a carrier molecule seems to be beneficial for some but not all isolation methods; and partially or fully quantitative recovery of RNA is observed from increasing volumes of plasma and cerebrospinal fluid.

  3. 高温弱碱稀盐法从啤酒废酵母中提取RNA%Extracting RNA from spent brewer's yeast by the new technology rare salt of high temperature of weak base

    Institute of Scientific and Technical Information of China (English)

    何娟; 詹自力; 夏萍; 曹颖; 卢奎


    采用盐法与碱法相结合破壁的方法,即高温弱碱稀盐法从啤酒废酵母中提取核糖核酸(RNA).正交试验确定的最佳工艺条件为:抽提温度100 ℃,w(NaCl)=6%,抽提时间90 min,pH=8.在此条件下,RNA收率可达8.01%,较传统工艺收率有显著提高.

  4. Automated microfluidic DNA/RNA extraction with both disposable and reusable components (United States)

    Kim, Jungkyu; Johnson, Michael; Hill, Parker; Sonkul, Rahul S.; Kim, Jongwon; Gale, Bruce K.


    An automated microfluidic nucleic extraction system was fabricated with a multilayer polydimethylsiloxane (PDMS) structure that consists of sample wells, microvalves, a micropump and a disposable microfluidic silica cartridge. Both the microvalves and micropump structures were fabricated in a single layer and are operated pneumatically using a 100 µm PDMS membrane. To fabricate the disposable microfluidic silica cartridge, two-cavity structures were made in a PDMS replica to fit the stacked silica membranes. A handheld controller for the microvalves and pumps was developed to enable system automation. With purified ribonucleic acid (RNA), whole blood and E. coli samples, the automated microfluidic nucleic acid extraction system was validated with a guanidine-based solid phase extraction procedure. An extraction efficiency of ~90% for deoxyribonucleic acid (DNA) and ~54% for RNA was obtained in 12 min from whole blood and E. coli samples, respectively. In addition, the same quantity and quality of extracted DNA was confirmed by polymerase chain reaction (PCR) amplification. The PCR also presented the appropriate amplification and melting profiles. Automated, programmable fluid control and physical separation of the reusable components and the disposable components significantly decrease the assay time and manufacturing cost and increase the flexibility and compatibility of the system with downstream components.

  5. Entropy-based model for miRNA isoform analysis.

    Directory of Open Access Journals (Sweden)

    Shengqin Wang

    Full Text Available MiRNAs have been widely studied due to their important post-transcriptional regulatory roles in gene expression. Many reports have demonstrated the evidence of miRNA isoform products (isomiRs in high-throughput small RNA sequencing data. However, the biological function involved in these molecules is still not well investigated. Here, we developed a Shannon entropy-based model to estimate isomiR expression profiles of high-throughput small RNA sequencing data extracted from miRBase webserver. By using the Kolmogorov-Smirnov statistical test (KS test, we demonstrated that the 5p and 3p miRNAs present more variants than the single arm miRNAs. We also found that the isomiR variant, except the 3' isomiR variant, is strongly correlated with Minimum Free Energy (MFE of pre-miRNA, suggesting the intrinsic feature of pre-miRNA should be one of the important factors for the miRNA regulation. The functional enrichment analysis showed that the miRNAs with high variation, particularly the 5' end variation, are enriched in a set of critical functions, supporting these molecules should not be randomly produced. Our results provide a probabilistic framework for miRNA isoforms analysis, and give functional insights into pre-miRNA processing.

  6. Evaluation of RNA extraction methods in rice and their application in expression analysis of resistance genes against Magnaporthe oryzae

    Directory of Open Access Journals (Sweden)

    Parisa Azizi


    Full Text Available Extraction of RNA of high quality and integrity is essential for gene expression studies and all downstream RNA-based techniques. The leaves of 16 merit Malaysian rice varieties were used to isolate total RNA using five different methods. The quantity, quality and integrity of extracted RNA were confirmed using three different means. The ratios of A260/280 ranged from 2.12 to 2.20. Electrophoresis (1.5% agarose gel was performed, illustrating intact and sharp bands representing the 28S, 18S, 5.8S and 5S ribosomal subunits of RNA, presenting intact RNA. RNA quality was verified using semi-quantitative polymerase chain reaction (sqPCR. The objective of this study was to identify different genes involved in the resistance of rice plants using high-quality RNA extracted 31 h after inoculation of Magnaporthe oryzae pathotype P7.2. The expression levels of eight blast resistance genes, Pikh, Pib, Pita, Pi21, Pi9, Os11gRGA8, OsWRKY22 and OsWRKY45, were evaluated by real-time PCR (RT-PCR. Real-time PCR was performed to identify candidate genes using RNA extracted by the TRIzol method, which showed the highest score compared with other methods in terms of RNA quantity, purity and integrity. In addition, the results of real-time PCR confirmed that the up-regulation of seven blast resistance genes may confer stronger resistance for the MR 276 variety against M. oryzae pathotype P7.2.

  7. RNA preservation agents and nucleic acid extraction method bias perceived bacterial community composition.

    Directory of Open Access Journals (Sweden)

    Ann McCarthy

    Full Text Available Bias is a pervasive problem when characterizing microbial communities. An important source is the difference in lysis efficiencies of different populations, which vary depending on the extraction protocol used. To avoid such biases impacting comparisons between gene and transcript abundances in the environment, the use of one protocol that simultaneously extracts both types of nucleic acids from microbial community samples has gained popularity. However, knowledge regarding tradeoffs to combined nucleic acid extraction protocols is limited, particularly regarding yield and biases in the observed community composition. Here, we evaluated a commercially available protocol for simultaneous extraction of DNA and RNA, which we adapted for freshwater microbial community samples that were collected on filters. DNA and RNA yields were comparable to other commonly used, but independent DNA and RNA extraction protocols. RNA protection agents benefited RNA quality, but decreased DNA yields significantly. Choice of extraction protocol influenced the perceived bacterial community composition, with strong method-dependent biases observed for specific phyla such as the Verrucomicrobia. The combined DNA/RNA extraction protocol detected significantly higher levels of Verrucomicrobia than the other protocols, and those higher numbers were confirmed by microscopic analysis. Use of RNA protection agents as well as independent sequencing runs caused a significant shift in community composition as well, albeit smaller than the shift caused by using different extraction protocols. Despite methodological biases, sample origin was the strongest determinant of community composition. However, when the abundance of specific phylogenetic groups is of interest, researchers need to be aware of the biases their methods introduce. This is particularly relevant if different methods are used for DNA and RNA extraction, in addition to using RNA protection agents only for RNA

  8. Comparative evaluation of total RNA extraction methods in Theobroma cacao using shoot apical meristems. (United States)

    Silva, D V; Branco, S M J; Holanda, I S A; Royaert, S; Motamayor, J C; Marelli, J P; Corrêa, R X


    Theobroma cacao is a species of great economic importance with its beans used for chocolate production. The tree has been a target of various molecular studies. It contains many polyphenols, which complicate the extraction of nucleic acids with the extraction protocols requiring a large amount of plant material. These issues, therefore, necessitate the optimization of the protocols. The aim of the present study was to evaluate different methods for extraction of total RNA from shoot apical meristems of T. cacao 'CCN 51' and to assess the influence of storage conditions for the meristems on the extraction. The study also aimed to identify the most efficient protocol for RNA extraction using a small amount of plant material. Four different protocols were evaluated for RNA extraction using one shoot apical meristem per sample. Among these protocols, one that was more efficient was then tested to extract RNA using four different numbers of shoot apical meristems, subjected to three different storage conditions. The best protocol was tested for cDNA amplification using reverse transcription-polymerase chain reaction; the cDNA quality was determined to be satisfactory for molecular analyses. The study revealed that with the best RNA extraction protocol, one shoot apical meristem was sufficient for extraction of high-quality total RNA. The results obtained might enable advances in genetic analyses and molecular studies using reduced amount of plant material.

  9. A modified protocol for RNA extraction from different peach tissues suitable for gene isolation and real-time PCR analysis. (United States)

    Tong, Zhaoguo; Qu, Shenchun; Zhang, Jiyu; Wang, Fei; Tao, Jianmin; Gao, Zhihong; Zhang, Zhen


    RNA extraction is the first step in the study of gene isolation and expression. However, it is difficult to extract high quantity and quality RNA from tissues containing large quantities of polysaccharides and polyphenols. Peach (Prunus persica), in addition to containing high levels of polysaccharides and polyphenols, is a challenging starting material for RNA isolation using a single method because of different amounts of those substances in diverse tissues. Based on three reported methods, we developed a modified RNA isolation protocol to solve this problem, leading to high quality and quantity of total RNA from peach mesocarp tissues of fruits which were sampled from all developmental stages and different storage periods, as well as from other tissues including flowers, leaves, stems, and roots. With our modified method, 28-650 μg of total RNA was routinely obtained from per gram of fresh material, gave at least a 1.16-fold improvement by compared with those isolated by other seven methods. The RNA extracts were successfully used in downstream applications such as RT-PCR, RACE, and real-time PCR.

  10. qPCR based mRNA quality score show intact mRNA after heat stabilization

    Directory of Open Access Journals (Sweden)

    Oskar Karlsson


    Full Text Available Analysis of multiple analytes from biological samples can be challenging as different analytes require different preservation measures. Heat induced enzymatic inactivation is an efficient way to preserve proteins and their modifications in biological samples but RNA quality, as measured by RIN value, has been a concern in such samples. Here, we investigate the effect of heat stabilization compared with standard snap freezing on RNA quality using two RNA extraction protocols, QiaZol with and without urea pre-solubilization, and two RNA quality measurements: RIN value, as defined by the Agilent Bioanalyzer, and an alternative qPCR based method. DNA extraction from heat stabilized brain samples was also examined. The snap frozen samples had RIN values about 1 unit higher than heat stabilized samples for the direct QiaZol extraction but equal with stabilized samples using urea pre-solubilization. qPCR based RNA quality measurement showed no difference in quality between snap frozen and heat inactivated samples. The probable explanation for this discrepancy is that the RIN value is an indirect measure based on rRNA, while the qPCR score is based on actual measurement of mRNA quality. The DNA yield from heat stabilized brain tissue samples was significantly increased, compared to the snap frozen tissue, without any effects on purity or quality. Hence, heat stabilization of tissues opens up the possibility for a two step preservation protocol, where proteins and their modifications can be preserved in the first heat based step, while in a second step, using standard RNA preservation strategies, mRNA be preserved. This collection strategy will enable biobanking of samples where the ultimate analysis is not determined without loss of sample quality.

  11. Evidence for an RNA polymerization activity in axolotl and Xenopus egg extracts.

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    Hélène Pelczar

    Full Text Available We have previously reported a post-transcriptional RNA amplification observed in vivo following injection of in vitro synthesized transcripts into axolotl oocytes, unfertilized (UFE or fertilized eggs. To further characterize this phenomenon, low speed extracts (LSE from axolotl and Xenopus UFE were prepared and tested in an RNA polymerization assay. The major conclusions are: i the amphibian extracts catalyze the incorporation of radioactive ribonucleotide in RNase but not DNase sensitive products showing that these products correspond to RNA; ii the phenomenon is resistant to α-amanitin, an inhibitor of RNA polymerases II and III and to cordycepin (3'dAMP, but sensitive to cordycepin 5'-triphosphate, an RNA elongation inhibitor, which supports the existence of an RNA polymerase activity different from polymerases II and III; the detection of radiolabelled RNA comigrating at the same length as the exogenous transcript added to the extracts allowed us to show that iii the RNA polymerization is not a 3' end labelling and that iv the radiolabelled RNA is single rather than double stranded. In vitro cell-free systems derived from amphibian UFE therefore validate our previous in vivo results hypothesizing the existence of an evolutionary conserved enzymatic activity with the properties of an RNA dependent RNA polymerase (RdRp.

  12. Extraction of ribosomal RNA and genomic DNA from soil for studying the diversity of the indigenous bacterial community

    NARCIS (Netherlands)

    Duarte, G.F.; Rosado, A.S.; Keijzer-Wolters, A.C.; Elsas, van J.D.


    A method for the indirect (cell extraction followed by nucleic acid extraction) isolation of bacterial ribosomal RNA (rRNA) and genomic DNA from soil was developed. The protocol allowed for the rapid parallel extraction of genomic DNA as well as small and large ribosomal subunit RNA from four soils

  13. Screening for plant viruses by next generation sequencing using a modified double strand RNA extraction protocol with an internal amplification control. (United States)

    Kesanakurti, Prasad; Belton, Mark; Saeed, Hanaa; Rast, Heidi; Boyes, Ian; Rott, Michael


    The majority of plant viruses contain RNA genomes. Detection of viral RNA genomes in infected plant material by next generation sequencing (NGS) is possible through the extraction and sequencing of total RNA, total RNA devoid of ribosomal RNA, small RNA interference (RNAi) molecules, or double stranded RNA (dsRNA). Plants do not typically produce high molecular weight dsRNA, therefore the presence of dsRNA makes it an attractive target for plant virus diagnostics. The sensitivity of NGS as a diagnostic method demands an effective dsRNA protocol that is both representative of the sample and minimizes sample cross contamination. We have developed a modified dsRNA extraction protocol that is more efficient compared to traditional protocols, requiring reduced amounts of starting material, that is less prone to sample cross contamination. This was accomplished by using bead based homogenization of plant material in closed, disposable 50ml tubes. To assess the quality of extraction, we also developed an internal control by designing a real-time (quantitative) PCR (qPCR) assay that targets endornaviruses present in Phaseolus vulgaris cultivar Black Turtle Soup (BTS).

  14. Literature-based condition-specific miRNA-mRNA target prediction (United States)

    Oh, Minsik; Rhee, Sungmin; Moon, Ji Hwan; Chae, Heejoon; Lee, Sunwon; Kang, Jaewoo; Kim, Sun


    miRNAs are small non-coding RNAs that regulate gene expression by binding to the 3′-UTR of genes. Many recent studies have reported that miRNAs play important biological roles by regulating specific mRNAs or genes. Many sequence-based target prediction algorithms have been developed to predict miRNA targets. However, these methods are not designed for condition-specific target predictions and produce many false positives; thus, expression-based target prediction algorithms have been developed for condition-specific target predictions. A typical strategy to utilize expression data is to leverage the negative control roles of miRNAs on genes. To control false positives, a stringent cutoff value is typically set, but in this case, these methods tend to reject many true target relationships, i.e., false negatives. To overcome these limitations, additional information should be utilized. The literature is probably the best resource that we can utilize. Recent literature mining systems compile millions of articles with experiments designed for specific biological questions, and the systems provide a function to search for specific information. To utilize the literature information, we used a literature mining system, BEST, that automatically extracts information from the literature in PubMed and that allows the user to perform searches of the literature with any English words. By integrating omics data analysis methods and BEST, we developed Context-MMIA, a miRNA-mRNA target prediction method that combines expression data analysis results and the literature information extracted based on the user-specified context. In the pathway enrichment analysis using genes included in the top 200 miRNA-targets, Context-MMIA outperformed the four existing target prediction methods that we tested. In another test on whether prediction methods can re-produce experimentally validated target relationships, Context-MMIA outperformed the four existing target prediction methods. In

  15. On-Orbit DNA, RNA, and Protein Extraction Project (United States)

    National Aeronautics and Space Administration — Genova Engineering proposes to develop and demonstrate a toolset of discrete devices and extraction kits which will leverage existing on-orbit facilities and will...

  16. 玉米矮花叶病毒CP基因dsRNA的原核表达与分离%Prokaryotic Expression and Extraction of dsRNA Based on the CP Gene of Maize Dwarf Mosaic Virus

    Institute of Scientific and Technical Information of China (English)

    甘德芳; 张姣; 赵阳; 朱苏文; 程备久


    根据玉米矮花叶病毒CP基因序列设计特异性引物,RT-PCR扩增玉米矮花叶病毒CP基因特异性干涉片段,将干涉片段及pUCCRNAi载体分别用BamH I及Sal I双酶切,然后将干涉片段分别正反向插入pUC- CRNAi载体中,构建CP基因反向重复克隆载体pUCCRNAi+2 F.再利用Pst I-Sal I位点插入到L4440质粒中构建原核表达载体LMCP.利用IPTG进行诱导表达并对诱导表达条件进行优化.结果表明,经过IPTG诱导,LMCP在大肠杆菌HT115(DE3)菌株中可表达产生预期大小的核酸片段,经DNase I和RNase A消化处理,证实为dsRNA.同时IPTG浓度为0.4~0.6 mmol/L,诱导表达4 h,dsRNA的表达量最高.另外,溶解于ddH2O中的dsRNA稳定性要高于溶解在NaCl中的,且随着放置时间的延长,dsRNA将出现明显的降解.%MDMV CP gene fragments were amplified by RT-PCR from extracted MDMV mRNA. To prepare a hairpin RNA, MDMV CP gene fragments and the pUCCRNAi cloning vector were digested by BamH I-Sal I respectively, First,the BamH I-Sal I fragment from MDMV RNA was cloned in the positive orientation into pUCCRNAi to generate pUCCRNAi + F. And then, the other BamH I-Sal I fragment was cloned in the reverse orientation into Bgl II-Xho I digested pUCCRNAi + F to generate an inverted repeat sequence of pUCCRNAi + 2 F ( sense orientation fragment and antisense orientation fragment were separated by an intron). Thirdly, L4440 and pUCCRNAi + 2 F plasmids were digested with Pst I-Sal I and subsequently joined to generate LMCP. And the recombinant plasmid was induced by IPTG. The results showed that the expression product was the dsRNA by treating with RNase A or DNase I to remove single-stranded RNA or DNA, respectively. Meanwhile, an IPTG concentration of 0.4 ~ 0.6 mmol/L and induction time of 4 h was the most optimal expression condition. The stability of the dsRNA in ddH20 is higher than that of in NaCl, and the dsRNA appeares to he dissolved with the time extending.

  17. RNA extraction from plant tissues: the use of calcium to precipitate contaminating pectic sugars. (United States)

    Dal Cin, Valeriano; Danesin, Marcello; Rizzini, Fabio Massimo; Ramina, Angelo


    Several protocols and commercial kits are used for the extraction of nucleic acids from different plant tissues. Although there are several procedures available to remove sugars, which hinder the extraction of clean genomic DNA, there are few to assist with extraction of RNA. Those presently used include precipitations with ethylene glycol monobutyl ether or lithium chloride (LiCl), or centrifugation in cesium chloride (CsCl) gradients, but these generally either do not allow high recovery of RNA, are time consuming, rely on hazardous chemicals or need special equipment. Here we present the use of the simple cation, Ca2+, which has been tested and shown to be very efficient for the precipitation of high molecular weight pectic sugars during RNA extraction. Results are presented for different plant tissues, especially tissues of peach and apple fruits at varying ripening stages.

  18. Web-Based Information Extraction Technology

    Institute of Scientific and Technical Information of China (English)


    Information extraction techniques on the Web are the current research hotspot. Now many information extraction techniques based on different principles have appeared and have different capabilities. We classify the existing information extraction techniques by the principle of information extraction and analyze the methods and principles of semantic information adding, schema defining,rule expression, semantic items locating and object locating in the approaches. Based on the above survey and analysis,several open problems are discussed.

  19. Influence of DNA extraction on oral microbial profiles obtained via 16S rRNA gene sequencing

    Directory of Open Access Journals (Sweden)

    Loreto Abusleme


    Full Text Available Background and objective: The advent of next-generation sequencing has significantly facilitated characterization of the oral microbiome. Despite great efforts in streamlining the processes of sequencing and data curation, upstream steps required for amplicon library generation could still influence 16S rRNA gene-based microbial profiles. Among upstream processes, DNA extraction is a critical step that could represent a great source of bias. Accounting for bias introduced by extraction procedures is important when comparing studies that use different methods. Identifying the method that best portrays communities is also desirable. Accordingly, the aim of this study was to evaluate bias introduced by different DNA extraction procedures on oral microbiome profiles. Design: Four DNA extraction methods were tested on mock communities consisting of seven representative oral bacteria. Additionally, supragingival plaque samples were collected from seven individuals and divided equally to test two commonly used DNA extraction procedures. Amplicon libraries of the 16S rRNA gene were generated and sequenced via 454-pyrosequencing. Results: Evaluation of mock communities revealed that DNA yield and bacterial species representation varied with DNA extraction methods. Despite producing the lowest yield of DNA, a method that included bead beating was the only protocol capable of detecting all seven species in the mock community. Comparison of the performance of two commonly used methods (crude lysis and a chemical/enzymatic lysis+column-based DNA isolation on plaque samples showed no effect of extraction protocols on taxa prevalence but global community structure and relative abundance of individual taxa were affected. At the phylum level, the latter method improved the recovery of Actinobacteria, Bacteroidetes, and Spirochaetes over crude lysis. Conclusion: DNA extraction distorts microbial profiles in simulated and clinical oral samples, reinforcing the

  20. High-quality RNA extraction from the sea urchin Paracentrotus lividus embryos (United States)

    Ruocco, Nadia; Costantini, Susan; Zupo, Valerio; Romano, Giovanna; Ianora, Adrianna; Fontana, Angelo; Costantini, Maria


    The sea urchin Paracentrotus lividus (Lamarck, 1816) is a keystone herbivore in the Mediterranean Sea due to its ability to transform macroalgal-dominated communities into barren areas characterized by increased cover of bare substrates and encrusting coralline algae, reduced biodiversity and altered ecosystem functions. P. lividus is also an excellent animal model for toxicology, physiology and biology investigations having been used for more than a century as a model for embryological studies with synchronously developing embryos which are easy to manipulate and analyze for morphological aberrations. Despite its importance for the scientific community, the complete genome is still not fully annotated. To date, only a few molecular tools are available and a few Next Generation Sequencing (NGS) studies have been performed. Here we aimed at setting-up an RNA extraction method to obtain high quality and sufficient quantity of RNA for NGS from P. lividus embryos at the pluteus stage. We compared five different RNA extraction protocols from four different pools of plutei (500, 1000, 2500 and 5000 embryos): TRIzol®, and four widely-used Silica Membrane kits, GenElute™ Mammalian Total RNA Miniprep Kit, RNAqueous® Micro Kit, RNeasy® Micro Kit and Aurum™ Total RNA Mini Kit. The quantity of RNA isolated was evaluated using NanoDrop. The quality, considering the purity, was measured as A260/A280 and A260/230 ratios. The integrity was measured by RNA Integrity Number (RIN). Our results demonstrated that the most efficient procedures were GenElute, RNeasy and Aurum, producing a sufficient quantity of RNA for NGS. The Bioanalyzer profiles and RIN values revealed that the most efficient methods guaranteeing for RNA integrity were RNeasy and Aurum combined with an initial preservation in RNAlater. This research represents the first attempt to standardize a method for high-quality RNA extraction from sea urchin embryos at the pluteus stage, providing a new resource for this

  1. A novel approach on fluid dispensing for a DNA/RNA extraction chip package (United States)

    Xie, Ling; Premachandran, C. S.; Chew, Michelle; Yao, Qiang; Xu, Diao; Pinjala, D.


    Micro fluidic package with integrated reservoirs has been developed for DNA /RNA extraction application. A membrane based pump which consists of a reservoir to store reagents and a pin valve to control the fluid is developed to dispense the reagents into the chip. A programmable external actuator is fabricated to dispense the fluid from the membrane pump into the DNA chip. An elastic and high elongation thin rubber membrane is used to seal the membrane pump and at the same time prevent actuator from mixing with different reagents in the micro fluidic package. Break displacement during actuation of membrane pump sealing material is studied with different ratios of PDMS and other types of rubber materials. The fluid flow from the reservoir to the chip is controlled by a pin valve which is activated during the external actuation. A CFD simulation is performed to study the pumping action dusting the external actuation and is validated with experimental results.

  2. A microfluidic approach for high efficiency extraction of low molecular weight RNA. (United States)

    Vulto, Paul; Dame, Gregory; Maier, Urban; Makohliso, Solomzi; Podszun, Susann; Zahn, Peter; Urban, Gerald A


    The lack of sample pre-treatment concepts that are easily automatable, miniaturized and highly efficient for both small volumes and low target concentrations, is one of the key issues that block the road towards effective miniaturized diagnostic instruments. This paper presents a novel, highly efficient and simple method for low-molecular weight RNA extraction using electricity only. Cells are lysed by thermo-electric lysis and RNA is purified using a gel-electrophoretic purification step. The combination of the two steps in one integrated cartridge reduces the time frame between the two steps, thus protecting RNA from enzymatic degradation. A disposable chip solution is proposed using a novel dry film resist laminate technology that allows cheap, large-scale fabrication. The chip contains crucial microfluidic innovations that allow for a simple user interface, reproducible functioning and precise quantification. Phaseguides are invented that allow controlled spatial injection of gel, injection of sample and recovery of extracted RNA. A precise sample volume can be defined by integrating electrophoretic actuation electrodes in the microfluidic chamber. Electrolytic gas bubbles that are the result of constant-current actuation are driven out from the chip by the novel introduction of capillary bubble-expulsion techniques. The extraction approach and the functionality of the chip are demonstrated for Escherichia coli and Streptococcus thermophilus bacteria. Linear extraction behavior is obtained for transfer-messenger RNA down to one colony-forming unit per microlitre, or five colony-forming units per chip. The latter is an increase in extraction efficiency of a factor of 1000 with respect to the commercial extraction kit Ambion Ribopure. The chip shows particularly good performance for extraction of low-molecular weight RNA, thereby eliminating the need for large ribosomal RNA and DNA removal. RNA can be extracted in less than 11 min, being a speed-up of more than a

  3. Evaluation of a low-cost procedure for sampling, long-term storage, and extraction of RNA from blood for qPCR analyses

    DEFF Research Database (Denmark)

    Mærkedahl, Rasmus Baadsgaard; Frøkiær, Hanne; Lauritzen, Lotte


    Abstract Background: In large clinical trials, where RNA cannot be extracted immediately after sampling, preserving RNA in whole blood is a crucial initial step in obtaining robust qPCR data. The current golden standard for RNA preservation is costly and designed for time-consuming column-based RNA......-extraction. We investigated the use of lysis buffer for long-term storage of blood samples for qPCR analysis. Methods: Blood was collected from 13 healthy adults and diluted in MagMAX lysis/binding solution or PAXgene Blood RNA tubes and stored at -20 °C for 0, 1, or 4 months before RNA extraction...... by the matching method. RNA integrity, yield and purity were evaluated and the methods were compared by subsequent analyses of the gene expression levels of 18S, ACTB, IL1B, IL1RN, IL1R2, and PGK1 using qPCR. Results: The MagMAX system extracted 2.3-2.8 times more RNA per mL blood, with better performance...

  4. Evaluation of Six Methods for Extraction of Total RNA from Loquat

    Directory of Open Access Journals (Sweden)

    Zhang LING


    Full Text Available   Trizol extraction for polysaccharide-rich plant tissue was unsuitable for isolating total RNA from loquat fruit. CTAB-LiCl extraction was improved with pretreatment using washing buffer with 80% ethanol and 70% acetone. The RNA isolated by this protocol from different loquat fruit tissue at various development periods, was high in purity (A260/A280 ratio ranged from 1.81 to 1.99 and A260/A230 ratio was over 2.0, and high in yield.

  5. iDoRNA: An Interacting Domain-based Tool for Designing RNA-RNA Interaction Systems

    Directory of Open Access Journals (Sweden)

    Jittrawan Thaiprasit


    Full Text Available RNA-RNA interactions play a crucial role in gene regulation in living organisms. They have gained increasing interest in the field of synthetic biology because of their potential applications in medicine and biotechnology. However, few novel regulators based on RNA-RNA interactions with desired structures and functions have been developed due to the challenges of developing design tools. Recently, we proposed a novel tool, called iDoDe, for designing RNA-RNA interacting sequences by first decomposing RNA structures into interacting domains and then designing each domain using a stochastic algorithm. However, iDoDe did not provide an optimal solution because it still lacks a mechanism to optimize the design. In this work, we have further developed the tool by incorporating a genetic algorithm (GA to find an RNA solution with maximized structural similarity and minimized hybridized RNA energy, and renamed the tool iDoRNA. A set of suitable parameters for the genetic algorithm were determined and found to be a weighting factor of 0.7, a crossover rate of 0.9, a mutation rate of 0.1, and the number of individuals per population set to 8. We demonstrated the performance of iDoRNA in comparison with iDoDe by using six RNA-RNA interaction models. It was found that iDoRNA could efficiently generate all models of interacting RNAs with far more accuracy and required far less computational time than iDoDe. Moreover, we compared the design performance of our tool against existing design tools using forty-four RNA-RNA interaction models. The results showed that the performance of iDoRNA is better than RiboMaker when considering the ensemble defect, the fitness score and computation time usage. However, it appears that iDoRNA is outperformed by NUPACK and RNAiFold 2.0 when considering the ensemble defect. Nevertheless, iDoRNA can still be an useful alternative tool for designing novel RNA-RNA interactions in synthetic biology research. The source code of iDoRNA

  6. How Severely Is DNA Quantification Hampered by RNA Co-extraction? (United States)

    Sanchez, Ignacio; Remm, Matthieu; Frasquilho, Sonia; Betsou, Fay; Mathieson, William


    The optional RNase digest that is part of many DNA extraction protocols is often omitted, either because RNase is not provided in the kit or because users do not want to risk contaminating their laboratory. Consequently, co-eluting RNA can become a "contaminant" of unknown magnitude in a DNA extraction. We extracted DNA from liver, lung, kidney, and heart tissues and established that 28-52% of the "DNA" as assessed by spectrophotometry is actually RNA (depending on tissue type). Including an RNase digest in the extraction protocol reduced 260:280 purity ratios. Co-eluting RNA drives an overestimation of DNA yield when quantification is carried out using OD 260 nm spectrophotometry, or becomes an unquantified contaminant when spectrofluorometry is used for DNA quantification. This situation is potentially incompatible with the best practice guidelines for biobanks issued by organizations such as the International Society for Biological and Environmental Repositories, which state that biospecimens should be accurately characterized in terms of their identity, purity, concentration, and integrity. Consequently, we conclude that an RNase digest must be included in DNA extractions if pure DNA is required. We also discuss the implications of unquantified RNA contamination in DNA samples in the context of laboratory accreditation schemes.

  7. RNA-Based Vaccines in Cancer Immunotherapy. (United States)

    McNamara, Megan A; Nair, Smita K; Holl, Eda K


    RNA vaccines traditionally consist of messenger RNA synthesized by in vitro transcription using a bacteriophage RNA polymerase and template DNA that encodes the antigen(s) of interest. Once administered and internalized by host cells, the mRNA transcripts are translated directly in the cytoplasm and then the resulting antigens are presented to antigen presenting cells to stimulate an immune response. Alternatively, dendritic cells can be loaded with either tumor associated antigen mRNA or total tumor RNA and delivered to the host to elicit a specific immune response. In this review, we will explain why RNA vaccines represent an attractive platform for cancer immunotherapy, discuss modifications to RNA structure that have been developed to optimize mRNA vaccine stability and translational efficiency, and describe strategies for nonviral delivery of mRNA vaccines, highlighting key preclinical and clinical data related to cancer immunotherapy.

  8. RNA-Based Vaccines in Cancer Immunotherapy

    Directory of Open Access Journals (Sweden)

    Megan A. McNamara


    Full Text Available RNA vaccines traditionally consist of messenger RNA synthesized by in vitro transcription using a bacteriophage RNA polymerase and template DNA that encodes the antigen(s of interest. Once administered and internalized by host cells, the mRNA transcripts are translated directly in the cytoplasm and then the resulting antigens are presented to antigen presenting cells to stimulate an immune response. Alternatively, dendritic cells can be loaded with either tumor associated antigen mRNA or total tumor RNA and delivered to the host to elicit a specific immune response. In this review, we will explain why RNA vaccines represent an attractive platform for cancer immunotherapy, discuss modifications to RNA structure that have been developed to optimize mRNA vaccine stability and translational efficiency, and describe strategies for nonviral delivery of mRNA vaccines, highlighting key preclinical and clinical data related to cancer immunotherapy.

  9. Improving Griffith's protocol for co-extraction of microbial DNA and RNA in adsorptive soils

    DEFF Research Database (Denmark)

    Paulin, Mélanie Marie; Nicolaisen, Mette Haubjerg; Jacobsen, Carsten Suhr


    -time PCR on both the RNA (after conversion to cDNA) and the DNA fraction of the extracts. Non-adsorptive soils were characterized by low clay content and/or high phosphate content, whereas adsorptive soils had clay contents above 20% and/or a strong presence of divalent Ca in combination with high p......Quantification of microbial gene expression is increasingly being used to study key functions in soil microbial communities, yet major limitations still exist for efficient extraction of nucleic acids, especially RNA for transcript analysis, from this complex matrix. We present an improved...... extraction protocol that was optimized by: i) including an adsorption-site competitor prior to cell lysis to decrease adsorption of nucleic acids to soil particles, and ii) optimizing the PEG concentration used for nucleic acid precipitation. The extraction efficiency was determined using quantitative real...

  10. Tissue extraction of DNA and RNA and analysis by the polymerase chain reaction. (United States)

    Jackson, D P; Lewis, F A; Taylor, G R; Boylston, A W; Quirke, P


    Several DNA extraction techniques were quantitatively and qualitatively compared using both fresh and paraffin wax embedded tissue and their suitability investigated for providing DNA and RNA for the polymerase chain reaction (PCR). A one hour incubation with proteinase K was the most efficient DNA extraction procedure for fresh tissue. For paraffin wax embedded tissue a five day incubation with proteinase K was required to produce good yields of DNA. Incubation with sodium dodecyl sulphate produced very poor yields, while boiling produced 20% as much DNA as long enzyme digestion. DNA extracted by these methods was suitable for the PCR amplification of a single copy gene. Proteinase K digestion also produced considerable amounts of RNA which has previously been shown to be suitable for PCR analysis. A delay before fixation had no effect on the amount of DNA obtained while fixation in Carnoy's reagent results in a much better preservation of DNA than formalin fixation, allowing greater yields to be extracted.

  11. Chemical and structural effects of base modifications in messenger RNA (United States)

    Harcourt, Emily M.; Kietrys, Anna M.; Kool, Eric T.


    A growing number of nucleobase modifications in messenger RNA have been revealed through advances in detection and RNA sequencing. Although some of the biochemical pathways that involve modified bases have been identified, research into the world of RNA modification -- the epitranscriptome -- is still in an early phase. A variety of chemical tools are being used to characterize base modifications, and the structural effects of known base modifications on RNA pairing, thermodynamics and folding are being determined in relation to their putative biological roles.

  12. Pattern Based Term Extraction Using ACABIT System

    CERN Document Server

    Takeuchi, Koichi; Koyama, Teruo; Daille, Béatrice; Romary, Laurent


    In this paper, we propose a pattern-based term extraction approach for Japanese, applying ACABIT system originally developed for French. The proposed approach evaluates termhood using morphological patterns of basic terms and term variants. After extracting term candidates, ACABIT system filters out non-terms from the candidates based on log-likelihood. This approach is suitable for Japanese term extraction because most of Japanese terms are compound nouns or simple phrasal patterns.

  13. A modular method for the extraction of DNA and RNA, and the separation of DNA pools from diverse environmental sample types

    DEFF Research Database (Denmark)

    Lever, Mark; Torti, Andrea; Eickenbusch, Philip


    A method for the extraction of nucleic acids from a wide range of environmental samples was developed. This method consists of several modules, which can be individually modified to maximize yields in extractions of DNA and RNA or separations of DNA pools. Modules were designed based on elaborate......'s oceans and the deepest borehole ever studied by scientific ocean drilling. Extraction yields of DNA and RNA are higher than with widely used commercial kits, indicating an advantage to optimizing extraction procedures to match specific sample characteristics. The ability to separate soluble extracellular...... tests, in which permutations of all nucleic acid extraction steps were compared. The final modular protocol is suitable for extractions from igneous rock, air, water, and sediments. Sediments range from high-biomass, organic rich coastal samples to samples from the most oligotrophic region of the world...

  14. RNA structural motif recognition based on least-squares distance. (United States)

    Shen, Ying; Wong, Hau-San; Zhang, Shaohong; Zhang, Lin


    RNA structural motifs are recurrent structural elements occurring in RNA molecules. RNA structural motif recognition aims to find RNA substructures that are similar to a query motif, and it is important for RNA structure analysis and RNA function prediction. In view of this, we propose a new method known as RNA Structural Motif Recognition based on Least-Squares distance (LS-RSMR) to effectively recognize RNA structural motifs. A test set consisting of five types of RNA structural motifs occurring in Escherichia coli ribosomal RNA is compiled by us. Experiments are conducted for recognizing these five types of motifs. The experimental results fully reveal the superiority of the proposed LS-RSMR compared with four other state-of-the-art methods.

  15. Total RNA extraction from strawberry tree (Arbutus unedo and several other woody-plants

    Directory of Open Access Journals (Sweden)

    Zamboni A


    Full Text Available Studies of plant gene expression today need pure preparations of high-yielding undegraded RNA. This is not easily accomplished when working with plants and tissues like strawberry tree (Arbutus unedo leaves that accumulate large amounts of polysaccharides and polyphenolic compounds, which co-purify with RNA. An improved leaf-tissue protocol developed for gene expression studies on Arbutus unedo yields for the first time a purity of RNA extract that makes possible cDNA synthesis and qPCR analysis in this plant species. When tested on material considered recalcitrant (leaves, roots, fruit flesh, fruit peel and styles from Pyrus communis, Prunus avium, Prunus persica and Cydonia oblonga, the method was able to extract RNA with good yield and high purity. This scalable, phenol-free, fast and easy-to-use RNA extraction protocol is effective on Arbutus unedo leaves as well as on awide range of different species and tissues, thus resulting particularly useful for gene expression analysis in non-model species for molecular biology.

  16. Extraction of High Quality RNA from Cannabis sativa Bast Fibres: A Vademecum for Molecular Biologists

    Directory of Open Access Journals (Sweden)

    Gea Guerriero


    Full Text Available In plants there is no universal protocol for RNA extraction, since optimizations are required depending on the species, tissues and developmental stages. Some plants/tissues are rich in secondary metabolites or synthesize thick cell walls, which hinder an efficient RNA extraction. One such example is bast fibres, long extraxylary cells characterized by a thick cellulosic cell wall. Given the economic importance of bast fibres, which are used in the textile sector, as well as in biocomposites as green substitutes of glass fibres, it is desirable to better understand their development from a molecular point of view. This knowledge favours the development of biotechnological strategies aimed at improving specific properties of bast fibres. To be able to perform high-throughput analyses, such as, for instance, transcriptomics of bast fibres, RNA extraction is a crucial and limiting step. We here detail a protocol enabling the rapid extraction of high quality RNA from the bast fibres of textile hemp, Cannabis sativa L., a multi-purpose fibre crop standing in the spotlight of research.

  17. Theoretical analysis of noncanonical base pairing interactions in RNA molecules

    Indian Academy of Sciences (India)

    Dhananjay Bhattacharyya; Siv Chand Koripella; Abhijit Mitra; Vijay Babu Rajendran; Bhabdyuti Sinha


    Noncanonical base pairs in RNA have strong structural and functional implications but are currently not considered for secondary structure predictions. We present results of comparative ab initio studies of stabilities and interaction energies for the three standard and 24 selected unusual RNA base pairs reported in the literature. Hydrogen added models of isolated base pairs, with heavy atoms frozen in their ‘away from equilibrium’ geometries, built from coordinates extracted from NDB, were geometry optimized using HF/6-31G** basis set, both before and after unfreezing the heavy atoms. Interaction energies, including BSSE and deformation energy corrections, were calculated, compared with respective single point MP2 energies, and correlated with occurrence frequencies and with types and geometries of hydrogen bonding interactions. Systems having two or more N-H…O/N hydrogen bonds had reasonable interaction energies which correlated well with respective occurrence frequencies and highlighted the possibility of some of them playing important roles in improved secondary structure prediction methods. Several of the remaining base pairs with one N-H…O/N and/or one C-H…O/N interactions respectively, had poor interaction energies and negligible occurrences. High geometry variations on optimization of some of these were suggestive of their conformational switch like characteristics.

  18. miRTex: A Text Mining System for miRNA-Gene Relation Extraction. (United States)

    Li, Gang; Ross, Karen E; Arighi, Cecilia N; Peng, Yifan; Wu, Cathy H; Vijay-Shanker, K


    MicroRNAs (miRNAs) regulate a wide range of cellular and developmental processes through gene expression suppression or mRNA degradation. Experimentally validated miRNA gene targets are often reported in the literature. In this paper, we describe miRTex, a text mining system that extracts miRNA-target relations, as well as miRNA-gene and gene-miRNA regulation relations. The system achieves good precision and recall when evaluated on a literature corpus of 150 abstracts with F-scores close to 0.90 on the three different types of relations. We conducted full-scale text mining using miRTex to process all the Medline abstracts and all the full-length articles in the PubMed Central Open Access Subset. The results for all the Medline abstracts are stored in a database for interactive query and file download via the website at Using miRTex, we identified genes potentially regulated by miRNAs in Triple Negative Breast Cancer, as well as miRNA-gene relations that, in conjunction with kinase-substrate relations, regulate the response to abiotic stress in Arabidopsis thaliana. These two use cases demonstrate the usefulness of miRTex text mining in the analysis of miRNA-regulated biological processes.

  19. Chinese Term Extraction Based on PAT Tree

    Institute of Scientific and Technical Information of China (English)

    ZHANG Feng; FAN Xiao-zhong; XU Yun


    A new method of automatic Chinese term extraction is proposed based on Patricia (PAT) tree. Mutual information is calculated based on prefix searching in PAT tree of domain corpus to estimate the internal associative strength between Chinese characters in a string. It can improve the speed of term candidate extraction largely compared with methods based on domain corpus directly. Common collocation suffix, prefix bank are constructed and term part of speech (POS) composing rules are summarized to improve the precision of term extraction. Experiment results show that the F-measure is 74.97 %.

  20. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development - Poster (United States)

    We examined the bacterial composition of chlorinated drinking water using 16S rRNA gene clone libraries derived from RNA and DNA extracted from twelve water samples collected in three different months (June, August, and September of 2007). Phylogenetic analysis of 1234 and 1117 ...

  1. A streamlined protocol for extracting RNA and genomic DNA from archived human blood and muscle. (United States)

    Majumdar, Gipsy; Vera, Santiago; Elam, Marshall B; Raghow, Rajendra


    We combined the TRIzol method of nucleic acid extraction with QIAamp columns to achieve coextraction of RNA and genomic DNA from peripheral blood mononuclear cells (PBMCs) and biopsied skeletal muscle, both stored at -80 °C for many months. Total RNA was recovered from the upper aqueous phase of TRIzol. The interphase and organic phases were precipitated with ethanol, digested with proteinase K, and filtered through QIAamp MinElute columns to recover DNA. The combined protocol yielded excellent quality and quantity of nucleic acids from archived human PBMCs and muscle and may be easily adapted for other tissues.

  2. Optimizing preservation protocols to extract high-quality RNA from different tissues of echinoderms for next-generation sequencing. (United States)

    Pérez-Portela, Rocío; Riesgo, Ana


    Transcriptomic information provides fundamental insights into biological processes. Extraction of quality RNA is a challenging step, and preservation and extraction protocols need to be adjusted in many cases. Our objectives were to optimize preservation protocols for isolation of high-quality RNA from diverse echinoderm tissues and to compare the utility of parameters as absorbance ratios and RIN values to assess RNA quality. Three different tissues (gonad, oesophagus and coelomocytes) were selected from the sea urchin Arbacia lixula. Solid tissues were flash-frozen and stored at -80 °C until processed. Four preservation treatments were applied to coelomocytes: flash freezing and storage at -80 °C, RNAlater and storage at -20 °C, preservation in TRIzol reagent and storage at -80 °C and direct extraction with TRIzol from fresh cells. Extractions of total RNA were performed with a modified TRIzol protocol for all tissues. Our results showed high values of RNA quantity and quality for all tissues, showing nonsignificant differences among them. However, while flash freezing was effective for solid tissues, it was inadequate for coelomocytes because of the low quality of the RNA extractions. Coelomocytes preserved in RNAlater displayed large variability in RNA integrity and insufficient RNA amount for further isolation of mRNA. TRIzol was the most efficient system for stabilizing RNA which resulted on high RNA quality and quantity. We did not detect correlation between absorbance ratios and RNA integrity. The best strategies for assessing RNA integrity was the visualization of 18S rRNA and 28S rRNA bands in agarose gels and estimation of RIN values with Agilent Bioanalyzer chips.

  3. Comparison of several methods of total RNA extraction for Candida tropicalis%几种提取热带假丝酵母总RNA方法比较

    Institute of Scientific and Technical Information of China (English)

    龙燕; 杨升


    Three methods including Trizol method, hot-phenol method, and Yeast RNA kit method were used for the total RNA extraction from Candida tropicalis. Based on total RNA concentration and purity, these methods were compared for their effects on total RNA extraction from Candida tropicalis. The results show the Yeast RNA kit method is the most effective and convenient method for total RNA extraction from Candida tropicalis.%选择Trizol法、热酚法、酵母RNA提取试剂盒法3种方法提取热带假丝酵母总RNA,通过对总RNA进行质量浓度和纯度分析,比较不同方法对热带假丝酵母总RNA提取的影响.结果表明:使用酵母RNA提取试剂盒法提取热带假丝酵母总RNA是较为有效且简便的方法.

  4. Development of a RNA extraction method from milk for gene expression study in the mammary gland of sheep. (United States)

    Mura, Maria Consuelo; Daga, Cinzia; Bodano, Sara; Paludo, Marta; Luridiana, Sebastiano; Pazzola, Michele; Dettori, Maria Luisa; Vacca, Giuseppe Massimo; Carcangiu, Vincenzo


    The aim of the study was to develop a reliable method for the RNA extraction from milk of Sarda sheep breed and to highlight if the extracted RNA can be used for expression study on mammary genes involved in milk fat synthesis using RT-qPCR. The main result is that a sample of 150 ml of milk provides an optimal amount of RNA (73.5 μg/ml). The highest RNA concentration has been found in the samples analysed within 4 h after collection. The RNA extracted was positively correlated to the number of somatic cells (P Ct value, for SREBPF1 gene of 26.8 ± 0.15. This research demonstrated that the high-quality of the RNA obtained is suited to use for studies of mammary genes expression in sheep, avoiding any damage caused by mammary gland biopsy.

  5. Effective DNA/RNA co-extraction for analysis of microRNAs, mRNAs, and genomic DNA from formalin-fixed paraffin-embedded specimens.

    Directory of Open Access Journals (Sweden)

    Adam Kotorashvili

    Full Text Available BACKGROUND: Retrospective studies of archived human specimens, with known clinical follow-up, are used to identify predictive and prognostic molecular markers of disease. Due to biochemical differences, however, formalin-fixed paraffin-embedded (FFPE DNA and RNA have generally been extracted separately from either different tissue sections or from the same section by dividing the digested tissue. The former limits accurate correlation whilst the latter is impractical when utilizing rare or limited archived specimens. PRINCIPAL FINDINGS: For effective recovery of genomic DNA and total RNA from a single FFPE specimen, without splitting the proteinase-K digested tissue solution, we optimized a co-extraction method by using TRIzol and purifying DNA from the lower aqueous and RNA from the upper organic phases. Using a series of seven different archived specimens, we evaluated the total amounts of genomic DNA and total RNA recovered by our TRIzol-based co-extraction method and compared our results with those from two commercial kits, the Qiagen AllPrep DNA/RNA FFPE kit, for co-extraction, and the Ambion RecoverAll™ Total Nucleic Acid Isolation kit, for separate extraction of FFPE-DNA and -RNA. Then, to accurately assess the quality of DNA and RNA co-extracted from a single FFPE specimen, we used qRT-PCR, gene expression profiling and methylation assays to analyze microRNAs, mRNAs, and genomic DNA recovered from matched fresh and FFPE MCF10A cells. These experiments show that the TRIzol-based co-extraction method provides larger amounts of FFPE-DNA and -RNA than the two other methods, and particularly provides higher quality microRNAs and genomic DNA for subsequent molecular analyses. SIGNIFICANCE: We determined that co-extraction of genomic DNA and total RNA from a single FFPE specimen is an effective recovery approach to obtain high-quality material for parallel molecular and high-throughput analyses. Our optimized approach provides the option of

  6. Classification and predictions of RNA pseudoknots based on topological invariants (United States)

    Vernizzi, Graziano; Orland, Henri; Zee, A.


    We propose a new topological characterization of ribonucleic acid (RNA) secondary structures with pseudoknots based on two topological invariants. Starting from the classic arc representation of RNA secondary structures, we consider a model that couples both (i) the topological genus of the graph and (ii) the number of crossing arcs of the corresponding primitive graph. We add a term proportional to these topological invariants to the standard free energy of the RNA molecule, thus obtaining a novel free-energy parametrization that takes into account the abundance of topologies of RNA pseudoknots observed in RNA databases.

  7. Multiple chromatographic peaks of phenylalanyl-tRNA associated with spontaneous hydrolysis of Y base during isolation. (United States)

    Mushinski, J F; Marini, M


    In contrast to the single phenylalanyl-tRNA found in normal cells, some tumours are known to have more than one phenylalanine isoacceptor. However, during certain steps in tRNA isolation from normal or tumour tissue, additional chromatographic peaks can be artificially produced which may be confused with the tumour-specific Phe-tRNA. Such procedures as extraction with unbuffered phenol and unbuffered gel filtration chromatography appear to produce adventitious isoacceptors by hydrolysis of Y base.

  8. Extraction of mRNA from coagulated horse blood and analysis of inflammation-related cytokine responses to coagulation

    DEFF Research Database (Denmark)

    Bovbjerg, Kirsten Katrine Lindegaard; Heegaard, Peter M. H.; Skovgaard, Kerstin


    .5-9.2), comparable with that purified by standard methods from stabilized blood. Cytokine mRNA expression was assessed by reverse transcribed quantitative real time PCR and it was found that 24-hour clotting led to a significant increase in the concentrations of mRNA of the pro-inflammatory cytokines interleukin-1β......Coagulated blood is a rich source of mRNA that allows the study of the regulation of expression of cytokine and other genes. However, while several methods are available for isolation of RNA from whole blood and tissues, protocols for purification of mRNA from clotted blood are not generally...... available. Here, a protocol for RNA extraction from highly clotted blood was optimized and the regulation of a number of cytokine genes compared to stabilized blood was studied. Whole blood samples from 10 clinically healthy horses were incubated for 24 hours at 37°C and RNA was extracted from...

  9. Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction. (United States)

    Patel, Palak G; Selvarajah, Shamini; Boursalie, Suzanne; How, Nathan E; Ejdelman, Joshua; Guerard, Karl-Philippe; Bartlett, John M; Lapointe, Jacques; Park, Paul C; Okello, John B A; Berman, David M


    Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

  10. Development of a Nucleic Acid Extraction Procedure for Simultaneous Recovery of DNA and RNA from Diverse Microbes in Water

    Directory of Open Access Journals (Sweden)

    Vincent R. Hill


    Full Text Available Drinking and environmental water samples contain a diverse array of constituents that can interfere with molecular testing techniques, especially when large volumes of water are concentrated to the small volumes needed for effective molecular analysis. In this study, a suite of enteric viruses, bacteria, and protozoan parasites were seeded into concentrated source water and finished drinking water samples, in order to investigate the relative performance of nucleic acid extraction techniques for molecular testing. Real-time PCR and reverse transcription-PCR crossing threshold (CT values were used as the metrics for evaluating relative performance. Experimental results were used to develop a guanidinium isothiocyanate-based lysis buffer (UNEX buffer that enabled effective simultaneous extraction and recovery of DNA and RNA from the suite of study microbes. Procedures for bead beating, nucleic acid purification, and PCR facilitation were also developed and integrated in the protocol. The final lysis buffer and sample preparation procedure was found to be effective for a panel of drinking water and source water concentrates when compared to commercial nucleic acid extraction kits. The UNEX buffer-based extraction protocol enabled PCR detection of six study microbes, in 100 L finished water samples from four drinking water treatment facilities, within three CT values (i.e., within 90% difference of the reagent-grade water control. The results from this study indicate that this newly formulated lysis buffer and sample preparation procedure can be useful for standardized molecular testing of drinking and environmental waters.

  11. PSRna: Prediction of small RNA secondary structures based on reverse complementary folding method. (United States)

    Li, Jin; Xu, Chengzhen; Wang, Lei; Liang, Hong; Feng, Weixing; Cai, Zhongxi; Wang, Ying; Cong, Wang; Liu, Yunlong


    Prediction of RNA secondary structures is an important problem in computational biology and bioinformatics, since RNA secondary structures are fundamental for functional analysis of RNA molecules. However, small RNA secondary structures are scarce and few algorithms have been specifically designed for predicting the secondary structures of small RNAs. Here we propose an algorithm named "PSRna" for predicting small-RNA secondary structures using reverse complementary folding and characteristic hairpin loops of small RNAs. Unlike traditional algorithms that usually generate multi-branch loops and 5[Formula: see text] end self-folding, PSRna first estimated the maximum number of base pairs of RNA secondary structures based on the dynamic programming algorithm and a path matrix is constructed at the same time. Second, the backtracking paths are extracted from the path matrix based on backtracking algorithm, and each backtracking path represents a secondary structure. To improve accuracy, the predicted RNA secondary structures are filtered based on their free energy, where only the secondary structure with the minimum free energy was identified as the candidate secondary structure. Our experiments on real data show that the proposed algorithm is superior to two popular methods, RNAfold and RNAstructure, in terms of sensitivity, specificity and Matthews correlation coefficient (MCC).

  12. 蟠桃果实总RNA提取方法的研究%RNA Extraction Method from Mature Flat Peach Fruits

    Institute of Scientific and Technical Information of China (English)

    史元敏; 何天明; 袁海英


    [Objective]The target of current research was to select and obtain a proper RNA extraction method from flat peach fruit. [ Method ] RNAplant plus, RNAisoTM, improved SDS method and improved CTAB method were used to isolate total RNA from flat peach fruits harvested at 100 days after full blossom. [Result]RNA extracted by two extraction kits had poor quality with contamination of the genome DNA; RNA extracted by improved SDS method contained large amounts of polysaccharide, while RNA extracted by the improved CTAB method had good integrity and the RNA bands were clear without degradation. RT - PCR and Northern blot test indicated that the extracted RNA could be used for die following molecular biology procedures. [ Conclusion ] The establishment of optimum RNA extraction mediod from flat peach fruits -improved CTAB method the base for the follow - up molecular biology study of fruit development.%[目的]筛选获得提取蟠桃果肉中总RNA的适宜方法.[方法]以盛花后100 d的蟠桃为材料,采用Transplant plus植物总RNA提取试剂提取法、RNAisoTM试剂盒法、改良SDS法、改良CTAB法等四种方法提取总RNA.[结果]两种试剂盒法提取的总RNA质量差,有严重的基因组污染;改良SDS法提取物中含有大量的多糖;采用改良CTAB法所得RNA完整性好,条带清晰无降解,无基因组污染.经过RT - PCR和Northern blot检测后,表明该RNA能够满足后续分子生物学操作的要求.[结论]蟠桃果实总RNA适宜提取方法-改良CTAB法的确定为后续果实发育分子生物学的研究奠定了基础.

  13. Genes and pathways affected by CAG-repeat RNA-based toxicity in Drosophila. (United States)

    Shieh, Shin-Yi; Bonini, Nancy M


    Spinocerebellar ataxia type 3 is one of the polyglutamine (polyQ) diseases, which are caused by a CAG-repeat expansion within the coding region of the associated genes. The CAG repeat specifies glutamine, and the expanded polyQ domain mutation confers dominant toxicity on the protein. Traditionally, studies have focused on protein toxicity in polyQ disease mechanisms. Recent findings, however, demonstrate that the CAG-repeat RNA, which encodes the toxic polyQ protein, also contributes to the disease in Drosophila. To provide insights into the nature of the RNA toxicity, we extracted brain-enriched RNA from flies expressing a toxic CAG-repeat mRNA (CAG100) and a non-toxic interrupted CAA/G mRNA repeat (CAA/G105) for microarray analysis. This approach identified 160 genes that are differentially expressed specifically in CAG100 flies. Functional annotation clustering analysis revealed several broad ontologies enriched in the CAG100 gene list, including iron ion binding and nucleotide binding. Intriguingly, transcripts for the Hsp70 genes, a powerful suppressor of polyQ and other human neurodegenerative diseases, were also upregulated. We therefore tested and showed that upregulation of heat shock protein 70 mitigates CAG-repeat RNA toxicity. We then assessed whether other modifiers of the pathogenic, expanded Ataxin-3 polyQ protein could also modify the CAG-repeat RNA toxicity. This approach identified the co-chaperone Tpr2, the transcriptional regulator Dpld, and the RNA-binding protein Orb2 as modifiers of both polyQ protein toxicity and CAG-repeat RNA-based toxicity. These findings suggest an overlap in the mechanisms of RNA and protein-based toxicity, providing insights into the pathogenicity of the RNA in polyQ disease.

  14. Magnetic Resonance Spectroscopy of siRNA-Based Cancer Therapy (United States)

    Penet, Marie-France; Chen, Zhihang; Mori, Noriko; Krishnamachary, Balaji; Bhujwalla, Zaver M.


    Small interfering RNA (siRNA) is routinely used as a biological tool to silence specific genes, and is under active investigation in cancer treatment strategies. Noninvasive magnetic resonance spectroscopy (MRS) provides the ability to assess the functional effects of siRNA-mediated gene silencing in cultured cancer cells, and following nanoparticle-based delivery in tumors in vivo. Here we describe the use of siRNA to downregulate choline kinase, a critical enzyme in choline phospholipid metabolism of cancer cells and tumors, and the use of 1H MRS of cells and 1H magnetic resonance spectroscopic imaging (MRSI) of tumors to assess the efficacy of the downregulation. PMID:26530913

  15. Cryptographic Protocols Based on Root Extracting

    DEFF Research Database (Denmark)

    Koprowski, Maciej

    In this thesis we design new cryptographic protocols, whose security is based on the hardness of root extracting or more speci cally the RSA problem. First we study the problem of root extraction in nite Abelian groups, where the group order is unknown. This is a natural generalization of the...... construction based on root extracting. As an example of this, we modify Cramer-Shoup signature scheme such that it becomes a genericm algorithm. We discuss then implementing it in RSA groups without the original restriction that the modulus must be a product of safe primes. It can also be implemented in class......,  providing a currently acceptable level of security. This allows us to propose the rst practical blind signature scheme provably secure, without relying on heuristics called random oracle model (ROM). We obtain the protocol for issuing blind signatures by implementing our modi ed Fischlin's signing algorithm...

  16. Molecular Beacon-Based MicroRNA Imaging During Neurogenesis. (United States)

    Lee, Jonghwan; Kim, Soonhag


    The fluorescence monitoring system for examining endogenous microRNA (miRNA) activity in cellular level provides crucial information on not only understanding a critical role of miRNA involving a variety of biological processes, but also evaluating miRNA expression patterns in a noninvasive manner. In this protocol, we report the details of a new procedure for a molecular beacon-based miRNA monitoring system, which includes the illustration scheme for miRNA detection strategy, exogenous miRNA detection, and measurement of endogenous miRNA expression level during neurogenesis. The fluorescence signal of miR-124a beacon quenched by BHQ2 was gradually recovered as increasing concentration of the miR-124a in tube. The functional work of miR-124a beacon was examined in intracellular environment, allowing for the internalization of the miR-124a beacon by lipofectamine, which resulted in activated fluorescent signals of the miR-124a beacon in the HeLa cells after the addition of synthetic miR-124a. The endogenous miR-124a expression level was detected by miR-124a beacon system during neurogenesis, showing brighter fluorescence intensity in cytoplasmic area of P19 cells after induction of neuronal differentiation by retinoic acid. The molecular beacon based-miRNA detection technique could be applicable to the simultaneous visualization of a variety of miRNA expression patterns using different fluorescence dyes. For the study of examining endogenous miRNA expression level using miRNA-beacon system, if cellular differentiation step is already prepared, transfection step of miR-124a beacon into P19 cells, and acquisition of activated fluorescence signal measured by confocal microscope can be conducted approximately within 6 h.

  17. Selective electromembrane extraction based on isoelectric point

    DEFF Research Database (Denmark)

    Huang, Chuixiu; Gjelstad, Astrid; Pedersen-Bjergaard, Stig


    For the first time, selective isolation of a target peptide based on the isoelectric point (pI) was achieved using a two-step electromembrane extraction (EME) approach with a thin flat membrane-based EME device. In this approach, step #1 was an extraction process, where both the target peptide...... angiotensin II antipeptide (AT2 AP, pI=5.13) and the matrix peptides (pI>5.13) angiotensin II (AT2), neurotensin (NT), angiotensin I (AT1) and leu-enkephalin (L-Enke) were all extracted as net positive species from the sample (pH 3.50), through a supported liquid membrane (SLM) of 1-nonanol diluted with 2......, and the target remained in the acceptor solution. The acceptor solution pH, the SLM composition, the extraction voltage, and the extraction time during the clean-up process (step #2) were important factors influencing the separation performance. An acceptor solution pH of 5.25 for the clean-up process slightly...

  18. Membrane-assisted culture of fungal mycelium on agar plates for RNA extraction and pharmacological analyses. (United States)

    Lange, Mario; Müller, Carolin; Peiter, Edgar


    Fungal mycelium grown in liquid culture is easy to harvest for RNA extraction and gene expression analyses, but liquid cultures often develop rather heterogeneously. In contrast, growth of fungal mycelium on agar plates is highly reproducible. However, this biological material cannot be harvested easily for downstream analyses. This article describes a PVDF (polyvinylidene difluoride) membrane-assisted agar plate culture method that enables the harvest of mycelium grown on agar plates. This culture method leads to a strongly reduced variation in gene expression between biological replicates and requires less growth space as compared with liquid cultures.

  19. A′-form RNA helices drive microtubule-based mRNA transport in Drosophila (United States)

    Bullock, Simon L.; Ringel, Inbal; Ish-Horowicz, David; Lukavsky, Peter J.


    Microtubule-based mRNA transport is widely used to restrict protein expression to specific regions in the cell, and has important roles in defining cell polarity, axis determination and for neuronal function. However, the structural basis of recognition of cis-acting mRNA localization signals by motor complexes is poorly understood. We have used NMR spectroscopy to describe the first tertiary structure of an RNA element responsible for mRNA transport. The Drosophila fs(1)K10 signal, which mediates transport by the dynein motor, forms a stem-loop with two double-stranded RNA helices adopting an unusual A′-form conformation with widened, major grooves reminiscent of those in B-form DNA. Structure determination of four mutant RNAs and extensive functional assays in Drosophila embryos indicate that the two spatially registered A′-form helices represent critical recognition sites for the transport machinery. Our study provides important insights into the basis for RNA cargo recognition and reveals a key biological function encoded by A′-form RNA conformation. PMID:20473315

  20. New short interfering RNA-based therapies for glomerulonephritis. (United States)

    Shimizu, Hideki; Fujita, Toshiro


    Current treatments for glomerulonephritis are not satisfactory, and the development of new therapies would be indispensable. Short interfering RNAs (siRNAs) are promising candidates for molecular therapy because of their strong and specific gene-silencing effects. Despite rapid progress in research into the therapeutic uses of siRNAs, however, many hurdles must be overcome before siRNA-based therapies can be brought to the clinic. Most in vivo studies of siRNA-based therapy have been limited to local administration or delivery to specific target organs, including the liver. Therapies based on siRNAs for patients with glomerulonephritis show promise, although tissue-specific protocols using siRNAs have not yet been established for this indication. This Review aims to provide an overview of the current challenges in siRNA-based therapy, primarily with respect to glomerular targeting. In addition, novel delivery approaches for glomerulus-targeted, siRNA-based therapies are described.

  1. [Influence of temperature on the preferential extraction of RNA polymerase I from hepatic nuclei of the rat]. (United States)

    Zoncheddu, A; Accomando, R; Pertica, M; Orunesu, M


    RNA polymerase I has been extracted from rat liver nuclei by three consecutive washings at 0 degrees C with a medium of relatively low ionic strength (0.15 M KCl) containing Mg++ rather than by incubating the organelles at 37 degrees C in the same medium, as originally proposed by Chesterton and Butterworth. The modified technique, which has the advantage of preventing a temperature-mediated conversion of form IB to IA, gives similar yields of RNA polymerase I and retains the capacity of preferentially extracting the enzyme with respect to the other forms of nuclear RNA polymerase.

  2. A simple and robust vector-based shRNA expression system used for RNA interference.

    Directory of Open Access Journals (Sweden)

    Xue-jun Wang

    Full Text Available BACKGROUND: RNA interference (RNAi mediated by small interfering RNAs (siRNAs or short hairpin RNAs (shRNAs has become a powerful genetic tool for conducting functional studies. Previously, vector-based shRNA-expression strategies capable of inducing RNAi in viable cells have been developed, however, these vector systems have some disadvantages, either because they were error-prone or cost prohibitive. RESULTS: In this report we described the development of a simple, robust shRNA expression system utilizing 1 long oligonucleotide or 2 short oligonucleotides for half the cost of conventional shRNA construction methods and with a >95% cloning success rate. The shRNA loop sequence and stem structure were also compared and carefully selected for better RNAi efficiency. Furthermore, an easier strategy was developed based on isocaudomers which permit rapid combination of the most efficient promoter-shRNA cassettes. Finally, using this method, the conservative target sites for hepatitis B virus (HBV knockdown were systemically screened and HBV antigen expression shown to be successfully suppressed in the presence of connected multiple shRNAs both in vitro and in vivo. CONCLUSION: This novel design describes an inexpensive and effective way to clone and express single or multiple shRNAs from the same vector with the capacity for potent and effective silencing of target genes.

  3. Design of RNA enzymes distinguishing a single base mutation in RNA. (United States)

    Koizumi, M; Hayase, Y; Iwai, S; Kamiya, H; Inoue, H; Ohtsuka, E


    RNA enzymes (ribozymes) which can cleave RNA by recognizing sequences of 9-15 bases are described. Substrates must contain UX (X = U, C or A). A ribozyme consisting of two oligoribonucleotides (19 mer and 15 mer) was shown to cleave a ribo 11 mer catalytically with Km and kcat values of 0.53 microM and 0.03 min-1, respectively. A non-cleavable substrate-ribozyme complex containing 2'-O-methylnucleoside was prepared and CD spectra were compared at different temperature. In order to obtain an efficient ribozyme, a one-strand RNA with a chain length of 37 was prepared. The ribozyme was shown to distinguish a single base mutation in mRNA's which were prepared by transcription of two synthetic DNA duplexes coding for positions 7-26 of c-Ha-ras protein. The mutant (Val-12) mRNA which had GUU was cleaved but the wild type mRNA which contained GGU was not changed, when treated by the ribozymes in the presence of Mg2+.

  4. Organocatalytic removal of formaldehyde adducts from RNA and DNA bases (United States)

    Karmakar, Saswata; Harcourt, Emily M.; Hewings, David S.; Lovejoy, Alexander F.; Kurtz, David M.; Ehrenschwender, Thomas; Barandun, Luzi J.; Roost, Caroline; Alizadeh, Ash A.; Kool, Eric T.


    Formaldehyde is universally used to fix tissue specimens, where it forms hemiaminal and aminal adducts with biomolecules, hindering the ability to retrieve molecular information. Common methods for removing these adducts involve extended heating, which can cause extensive degradation of nucleic acids, particularly RNA. Here, we show that water-soluble bifunctional catalysts (anthranilates and phosphanilates) speed the reversal of formaldehyde adducts of mononucleotides over standard buffers. Studies with formaldehyde-treated RNA oligonucleotides show that the catalysts enhance adduct removal, restoring unmodified RNA at 37 °C even when extensively modified, while avoiding the high temperatures that promote RNA degradation. Experiments with formalin-fixed, paraffin-embedded cell samples show that the catalysis is compatible with common RNA extraction protocols, with detectable RNA yields increased by 1.5-2.4-fold using a catalyst under optimized conditions and by 7-25-fold compared with a commercial kit. Such catalytic strategies show promise for general use in reversing formaldehyde adducts in clinical specimens.

  5. A novel method for RNA extraction from FFPE samples reveals significant differences in biomarker expression between orthotopic and subcutaneous pancreatic cancer patient-derived xenografts (United States)

    Brown, Mark; Maawy, Ali; Chang, Alexander; Lee, Jacqueline; Gharibi, Armen; Katz, Matthew H; Fleming, Jason; Hoffman, Robert M; Bouvet, Michael; Doebler, Robert; Kelber, Jonathan A


    Next-generation sequencing (NGS) can identify and validate new biomarkers of cancer onset, progression and therapy resistance. Substantial archives of formalin-fixed, paraffin-embedded (FFPE) cancer samples from patients represent a rich resource for linking molecular signatures to clinical data. However, performing NGS on FFPE samples is limited by poor RNA purification methods. To address this hurdle, we developed an improved methodology for extracting high-quality RNA from FFPE samples. By briefly integrating a newly-designed micro-homogenizing (mH) tool with commercially available FFPE RNA extraction protocols, RNA recovery is increased by approximately 3-fold while maintaining standard A260/A280 ratios and RNA quality index (RQI) values. Furthermore, we demonstrate that the mH-purified FFPE RNAs are longer and of higher integrity. Previous studies have suggested that pancreatic ductal adenocarcinoma (PDAC) gene expression signatures vary significantly under in vitro versus in vivo and in vivo subcutaneous versus orthotopic conditions. By using our improved mH-based method, we were able to preserve established expression patterns of KRas-dependency genes within these three unique microenvironments. Finally, expression analysis of novel biomarkers in KRas mutant PDAC samples revealed that PEAK1 decreases and MST1R increases by over 100-fold in orthotopic versus subcutaneous microenvironments. Interestingly, however, only PEAK1 levels remain elevated in orthotopically grown KRas wild-type PDAC cells. These results demonstrate the critical nature of the orthotopic tumor microenvironment when evaluating the clinical relevance of new biomarkers in cells or patient-derived samples. Furthermore, this new mH-based FFPE RNA extraction method has the potential to enhance and expand future FFPE-RNA-NGS cancer biomarker studies. PMID:27602776

  6. RNA is required for enzymatic conversion of glutamate to delta-aminolevulinate by extracts of Chlorella vulgaris. (United States)

    Weinstein, J D; Beale, S I


    Formation of delta-aminolevulinic acid (ALA) from glutamete catalyzed by a soluble extract from the unicellular green alga, Chlorella vulgaris, was abolished after incubation of the cell extract with bovine pancreatic ribonuclease A (RNase). Cell extract was prepared for the ALA formation assay by high-speed centrifugation and gel-filtration through Sephadex G-25 to remove insoluble and endogenous low-molecular-weight components. RNA hydrolysis products did not affect ALA formation, and RNase did not affect the ability of ATP and NADPH to serve as reaction substrates, indicating that the effect of RNase cannot be attributed to degradation of reaction substrates or transformation of a substrate or cofactor into an inhibitor. The effect of RNase was blocked by prior addition of placental RNase inhibitor (RNasin) to the cell extract, but RNasin did not reverse the effect of prior incubation of the cell extract with RNase, indicating that RNase does not act by degrading a component generated during the ALA-forming reaction, but instead degrades an essential component already present in active cell extract at the time the ALA-forming reaction is initiated. After inactivation of the cell extract by incubation with RNase, followed by administration of RNasin to block further RNase action, ALA-forming activity could be restored to a higher level than originally present by addition of a C. vulgaris tRNA-containing fraction isolated from an active ALA-forming preparation by phenol extraction and DEAE-cellulose chromatography. Baker's yeast tRNA, wheat germ tRNA, Escherichia coli tRNA, and E. coli tRNAglu type II were unable to reconstitute ALA-forming activity in RNase-treated cell extract, even though the cell extract was capable of catalyzing the charging of some of these RNAs with glutamate.

  7. 21 CFR 172.585 - Sugar beet extract flavor base. (United States)


    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Sugar beet extract flavor base. 172.585 Section 172... CONSUMPTION Flavoring Agents and Related Substances § 172.585 Sugar beet extract flavor base. Sugar beet...) Sugar beet extract flavor base is the concentrated residue of soluble sugar beet extractives from...

  8. Combinatorics of RNA Secondary Structures with Base Triples. (United States)

    Müller, Robert; Nebel, Markus E


    The structure of RNA has been the subject of intense research over the last decades due to its importance for the correct functioning of RNA molecules in biological processes. Hence, a large number of models for RNA folding and corresponding algorithms for structure prediction have been developed. However, previous models often only consider base pairs, although every base is capable of up to three edge-to-edge interactions with other bases. Recently, Höner zu Siederdissen et al. presented an extended model of RNA secondary structure, including base triples together with a folding algorithm-the first thermodynamics-based algorithm that allows the prediction of secondary structures with base triples. In this article, we investigate the search space processed by this new algorithm, that is, the combinatorics of extended RNA secondary structures with base triples. We present generalized definitions for structural motifs like hairpins, stems, bulges, or interior loops occurring in structures with base triples. Furthermore, we prove precise asymptotic results for the number of different structures (size of search space) and expectations for various parameters associated with structural motifs (typical shape of folding). Our analysis shows that the asymptotic number of secondary structures of size n increases exponentially to [Formula: see text] compared to the classic model by Stein and Waterman for which [Formula: see text] structures exist. A comparison with the classic model reveals large deviations in the expected structural appearance, too. The inclusion of base triples constitutes a significant refinement of the combinatorial model of RNA secondary structure, which, by our findings, is quantitatively characterized. Our results are of special theoretical interest, because a closer look at the numbers involved suggests that extended RNA secondary structures constitute a new combinatorial class not bijective with any other combinatorial objects studied so far.

  9. Synthetic Pre-miRNA-Based shRNA as Potent RNAi Triggers

    Directory of Open Access Journals (Sweden)

    Kazuya Terasawa


    Full Text Available RNA interference (RNAi is a powerful tool for studying gene function owing to the ease with which it can selectively silence genes of interest, and it has also attracted attention because of its potential for therapeutic applications. Chemically synthesized small interfering RNAs (siRNAs and DNA vector-based short hairpin RNAs (shRNAs are now widely used as RNAi triggers. In contrast to expressed shRNAs, the use of synthetic shRNAs is limited. Here we designed shRNAs modeled on a precursor microRNA (pre-miRNA and evaluated their biological activity. We demonstrated that chemically synthetic pre-miRNA-based shRNAs have more potent RNAi activity than their corresponding siRNAs and found that their antisense strands are more efficiently incorporated into the RNA-induced silencing complex. Although greater off-target effects and interferon responses were induced by shRNAs than by their corresponding siRNAs, these effects could be overcome by simply using a lower concentration or by optimizing and chemically modifying shRNAs similar to synthetic siRNAs. These are challenges for the future.

  10. Statistical feature extraction based iris recognition system

    Indian Academy of Sciences (India)



    Iris recognition systems have been proposed by numerous researchers using different feature extraction techniques for accurate and reliable biometric authentication. In this paper, a statistical feature extraction technique based on correlation between adjacent pixels has been proposed and implemented. Hamming distance based metric has been used for matching. Performance of the proposed iris recognition system (IRS) has been measured by recording false acceptance rate (FAR) and false rejection rate (FRR) at differentthresholds in the distance metric. System performance has been evaluated by computing statistical features along two directions, namely, radial direction of circular iris region and angular direction extending from pupil tosclera. Experiments have also been conducted to study the effect of number of statistical parameters on FAR and FRR. Results obtained from the experiments based on different set of statistical features of iris images show thatthere is a significant improvement in equal error rate (EER) when number of statistical parameters for feature extraction is increased from three to six. Further, it has also been found that increasing radial/angular resolution,with normalization in place, improves EER for proposed iris recognition system

  11. Lab-on-a-chip mRNA purification and reverse transcription via a solid-phase gene extraction technique. (United States)

    Nestorova, Gergana G; Hasenstein, Karl; Nguyen, Nam; DeCoster, Mark A; Crews, Niel D


    Extraction and purification of high quality RNA is a crucial initial step required for a variety of genomic assays. We report a solid phase gene extraction (SPGE) method for automated extraction, purification and reverse transcription of mRNA in a microfluidic device. This is performed using a 130 μm diameter stainless steel needle that is amino-linked to dT(15) oligonucleotides for selective hybridization of mRNA. By inserting this probe into the biological sample for only 30 seconds, mRNA is captured with high selectivity and a yield greater than 10 pg per mm of probe length. The probe is then inserted into a lab-on-a-chip device, where the bound poly-adenylated RNA is thermally released and immediately reverse transcribed for subsequent PCR amplification. The insertion of the probe into the microfluidic device is straightforward: the microchannel is formed with an elastomer (PDMS) that, when punctured, will seal around the probe. The specificity and RNA loading capacity of the probes were evaluated using conventional qPCR. This procedure was successfully used to extract, purify, and transcribe mRNA from rat glioblastoma cell spheroids in less than seven minutes. Analysis of the product confirmed that the SPGE technique selectively captures and inherently purifies high-quality mRNA directly from biological material with no need for additional pre-processing steps. Integrating this elegant sample preparation method into a complete lab-on-a-chip system will substantially enhance the speed and automation of mRNA assays for research and clinical diagnostics.

  12. Screening of total RNA extraction methods for RNA-sequences from different organs inZiziphus jujuba%适于转录组测序的枣不同器官总RNA提取方法筛选

    Institute of Scientific and Technical Information of China (English)

    魏琦琦; 冯延芝; 林青; 贾宝光; 张琳


    In order to obtain themethodsfor high-quality total RNA extractionfromlfower,fruit bearing branchandfruit inZiziphus jujuba, takingZ. jujuba cv.'Zhongqiusucui'asmaterials,and the RNA extraction effectswere compared using twokinds of total RNA extractionkits (Ambiogenand Autolab)andmodiifed CTABmethod based on the Autolab RNA extractionkit. Theresults showed thatOD260/OD280values of the RNA extracted by using the three RNA extractionmethods had no signiifcant differences, buta part of RNAs obtained by the two RNA extractionkitswere degraded. The RNAmass concentrationsfromlfower,fruit bearing branchandfruitwere 126, 284and 222 ng/μL by using the RNA extractionkit of Ambiogen; the RNAmass concentrationswere 307, 402and 266 ng/μL by using the RNA extractionkit of Autolab;and the RNAmass concentrationswere 401, 417and 296 ng/μL by using themodiifed CTABmethod based on the Autolab RNA extractionkit,respectively. Themodiifed CTABmethod couldgenerate high-integrity, high-concentrationand high-purity RNA that couldmeet therequest of transcriptome sequencing, comparedwith the two extractionkits.%为获得适用于转录组测序的枣花、结果枝和果实的总RNA提取方法,以中秋酥脆枣为材料,比较分析了Ambiogen和Autolab 2种总RNA提取试剂盒和基于Autolab试剂盒改良的CTAB法的总RNA的提取效果.结果表明:3种方法提取RNA的OD260/OD280差别不大,但用2种试剂盒提取的RNA有降解,其中Ambiogen试剂盒提取的花、结果枝和果实3个器官的RNA质量浓度分别为126、284和222 ng/μL;Autolab试剂盒提取的RNA质量浓度分别为307、402和266 ng/μL;基于Autolab试剂盒改良的CTAB法提取的RNA质量浓度分别为401、417和296 ng/μL.与2种试剂盒相比,基于Autolab试剂盒改良的CTAB法提取出来的RNA完整性好、纯度和浓度高,能够满足转录组测序的要求.

  13. Extracting Answer in QA System: Learning Based

    Directory of Open Access Journals (Sweden)

    Megha Mishra


    Full Text Available Converting questions to effective queries is crucial to open-domain question answering systems. In this paper, we present a web-based unsupervised learning approach for transforming a given natural-language question to an effective query. The method involves querying a search engine for web passages that contain the answer to the question, extracting patterns that characterize fine-grained classification for answers. Independent evaluation on a set of questions shows that the proposed approach outperforms a naive keyword based approach.

  14. Predicting siRNA efficacy based on multiple selective siRNA representations and their combination at score level (United States)

    He, Fei; Han, Ye; Gong, Jianting; Song, Jiazhi; Wang, Han; Li, Yanwen


    Small interfering RNAs (siRNAs) may induce to targeted gene knockdown, and the gene silencing effectiveness relies on the efficacy of the siRNA. Therefore, the task of this paper is to construct an effective siRNA prediction method. In our work, we try to describe siRNA from both quantitative and qualitative aspects. For quantitative analyses, we form four groups of effective features, including nucleotide frequencies, thermodynamic stability profile, thermodynamic of siRNA-mRNA interaction, and mRNA related features, as a new mixed representation, in which thermodynamic of siRNA-mRNA interaction is introduced to siRNA efficacy prediction for the first time to our best knowledge. And then an F-score based feature selection is employed to investigate the contribution of each feature and remove the weak relevant features. Meanwhile, we encode the siRNA sequence and existed empirical design rules as a qualitative siRNA representation. These two kinds of siRNA representations are combined to predict siRNA efficacy by supported Vector Regression (SVR) at score level. The experimental results indicate that our method may select the features with powerful discriminative ability and make the two kinds of siRNA representations work at full capacity. The prediction results also demonstrate that our method can outperform other popular siRNA efficacy prediction algorithms.

  15. A path-based measurement for human miRNA functional similarities using miRNA-disease associations (United States)

    Ding, Pingjian; Luo, Jiawei; Xiao, Qiu; Chen, Xiangtao


    Compared with the sequence and expression similarity, miRNA functional similarity is so important for biology researches and many applications such as miRNA clustering, miRNA function prediction, miRNA synergism identification and disease miRNA prioritization. However, the existing methods always utilized the predicted miRNA target which has high false positive and false negative to calculate the miRNA functional similarity. Meanwhile, it is difficult to achieve high reliability of miRNA functional similarity with miRNA-disease associations. Therefore, it is increasingly needed to improve the measurement of miRNA functional similarity. In this study, we develop a novel path-based calculation method of miRNA functional similarity based on miRNA-disease associations, called MFSP. Compared with other methods, our method obtains higher average functional similarity of intra-family and intra-cluster selected groups. Meanwhile, the lower average functional similarity of inter-family and inter-cluster miRNA pair is obtained. In addition, the smaller p-value is achieved, while applying Wilcoxon rank-sum test and Kruskal-Wallis test to different miRNA groups. The relationship between miRNA functional similarity and other information sources is exhibited. Furthermore, the constructed miRNA functional network based on MFSP is a scale-free and small-world network. Moreover, the higher AUC for miRNA-disease prediction indicates the ability of MFSP uncovering miRNA functional similarity.

  16. Small-interfering RNA (siRNA)-based functional micro- and nanostructures for efficient and selective gene silencing. (United States)

    Lee, Soo Hyeon; Chung, Bong Hyun; Park, Tae Gwan; Nam, Yoon Sung; Mok, Hyejung


    Because of RNA's ability to encode structure and functional information, researchers have fabricated diverse geometric structures from this polymer at the micro- and nanoscale. With their tunable structures, rigidity, and biocompatibility, novel two-dimensional and three-dimensional RNA structures can serve as a fundamental platform for biomedical applications, including engineered tissues, biosensors, and drug delivery vehicles. The discovery of the potential of small-interfering RNA (siRNA) has underscored the applications of RNA-based micro- and nanostructures in medicine. Small-interfering RNA (siRNA), synthetic double-stranded RNA consisting of approximately 21 base pairs, suppresses problematic target genes in a sequence-specific manner via inherent RNA interference (RNAi) processing. As a result, siRNA offers a potential strategy for treatment of many human diseases. However, due to inefficient delivery to cells and off-target effects, the clinical application of therapeutic siRNA has been very challenging. To address these issues, researchers have studied a variety of nanocarrier systems for siRNA delivery. In this Account, we describe several strategies for efficient siRNA delivery and selective gene silencing. We took advantage of facile chemical conjugation and complementary hybridization to design novel siRNA-based micro- and nanostructures. Using chemical crosslinkers and hydrophobic/hydrophilic polymers at the end of siRNA, we produced various RNA-based structures, including siRNA block copolymers, micelles, linear siRNA homopolymers, and microhydrogels. Because of their increased charge density and flexibility compared with conventional siRNA, these micro- and nanostructures can form polyelectrolyte complexes with poorly charged and biocompatible cationic carriers that are both more condensed and more homogenous than the complexes formed in other carrier systems. In addition, the fabricated siRNA-based structures are linked by cleavable disulfide

  17. Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR. (United States)

    Tatti, Enrico; McKew, Boyd A; Whitby, Corrine; Smith, Cindy J


    Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with 'end-point' PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included.

  18. Protein from the fraction remaining after RNA extraction is useful for proteomics but care must be exercised in its application. (United States)

    Yamaguchi, Hiromi; Hasegawa, Kiyoshi; Esumi, Mariko


    Simultaneous isolation of mRNA and proteins from a single small biopsy specimen can be useful for integrated omics studies. Here, we have improved the method for extracting protein from the fraction remaining after RNA isolation by TRIzol reagent, for application in protein and proteome analyses. Protein yield was reduced by half, but the patterns developed on 2D gels were equivalent to conventional urea extractions. Thus, although quantitative profiles of individual proteins were different from conventionally-isolated samples, overall profiles were similar. Therefore, this particular protein source is useful for proteomics but care must be exercised in its application.

  19. 土壤RNA提取方法研究进展%Progress of Researches on Extraction Methods of Soil RNA

    Institute of Scientific and Technical Information of China (English)

    刘华; 申天琳; 李学坤; 付合才; 戴九兰


    RNA plays an important role in studying environmental microbes because it can indicate the characteristics of the activated microorganism. The extraction of total RNA is the basis of real-time fluorescence quantitative PCR, northern hybridization analysis, cDNA library creation and other molecular biology studies. How to obtain a high yield and quality soil RNA is the critical step for the research of soil microbes. Therefore, several RNA isolation methods which can obtain high quality RNA from soil were summarized in this article, including strong denaturant method, Trizol method, CTAB method, SDS-phenol method, enzyme decomposition method and commercial RNA extraction kits. The various combinations of above methods for RNA isolation from soil were often used. Among the above methods, strong denaturant is good Rnase inhibitor, and CTAB can cause the protein denaturation. However, bead beating is the efficient physical lysis method. Compared with other methods, mechanical homogenization in Trizol is the most efficient for releasing microbial RNA. The different RNA extraction kits have their characteristics for different soils. Furthermore, the influence factors, application aspects, as well as the potentials and the limitations for the extraction of total RNA were also reviewed. Therefore, this work may provide the guidelines for RNA isolation and the choice of method to study soil microbes. Ref 56%总RNA提取是进行实时荧光定量PCR、Northern杂交分析及cDNA文库建立等分子生物学实验研究的基础.提取出高质量高产量的土壤RNA已成为研究土壤微生物的关键步骤.综述了常用的几种土壤RNA提取方法,包括强变性剂法、Trizol法、CTAB法、SDS-Phenol法、酶解法以及试剂盒等方法.提取过程中化学试剂的选择及配比需依据不同的土壤类型,其中Trizol法在提取总RNA时应用广泛,再结合适当的物理破碎及生物学方法,可得到理想的RNA.试剂盒法提取土壤RNA方

  20. SPIDIA-RNA: second external quality assessment for the pre-analytical phase of blood samples used for RNA based analyses.

    Directory of Open Access Journals (Sweden)

    Francesca Malentacchi

    Full Text Available One purpose of the EC funded project, SPIDIA, is to develop evidence-based quality guidelines for the pre-analytical handling of blood samples for RNA molecular testing. To this end, two pan-European External Quality Assessments (EQAs were implemented. Here we report the results of the second SPIDIA-RNA EQA. This second study included modifications in the protocol related to the blood collection process, the shipping conditions and pre-analytical specimen handling for participants. Participating laboratories received two identical proficiency blood specimens collected in tubes with or without an RNA stabilizer. For pre-defined specimen storage times and temperatures, laboratories were asked to perform RNA extraction from whole blood according to their usual procedure and to return extracted RNA to the SPIDIA facility for further analysis. These RNA samples were evaluated for purity, yield, integrity, stability, presence of interfering substances, and gene expression levels for the validated markers of RNA stability: FOS, IL1B, IL8, GAPDH, FOSB and TNFRSF10c. Analysis of the gene expression results of FOS, IL8, FOSB, and TNFRSF10c, however, indicated that the levels of these transcripts were significantly affected by blood collection tube type and storage temperature. These results demonstrated that only blood collection tubes containing a cellular RNA stabilizer allowed reliable gene expression analysis within 48 h from blood collection for all the genes investigated. The results of these two EQAs have been proposed for use in the development of a Technical Specification by the European Committee for Standardization.

  1. Building Extraction from LIDAR Based Semantic Analysis

    Institute of Scientific and Technical Information of China (English)

    YU Jie; YANG Haiquan; TAN Ming; ZHANG Guoning


    Extraction of buildings from LIDAR data has been an active research field in recent years. A scheme for building detection and reconstruction from LIDAR data is presented with an object-oriented method which is based on the buildings' semantic rules. Two key steps are discussed: how to group the discrete LIDAR points into single objects and how to establish the buildings' semantic rules. In the end, the buildings are reconstructed in 3D form and three common parametric building models (flat, gabled, hipped) are implemented.

  2. Advancing forensic RNA typing: On non-target secretions, a nasal mucosa marker, a differential co-extraction protocol and the sensitivity of DNA and RNA profiling. (United States)

    van den Berge, Margreet; Bhoelai, Bryan; Harteveld, Joyce; Matai, Anuska; Sijen, Titia


    The forensic identification of human body fluids and tissues by means of messenger RNA (mRNA) profiling is a long studied methodology that is increasingly applied to casework samples. Previously, we have described an mRNA multiplex system that targets blood, saliva, semen, menstrual secretion, vaginal mucosa and skin (Lindenbergh et al. and van den Berge et al.). In this study we consider various topics to improve this mRNA profiling system or its use and adapt the method accordingly. Bodily secretions that may be encountered at a crime scene whilst not targeted by the multiplex-id est nasal mucosa, sweat, tears, faeces and urine-were examined for false positive signals. The results prompted us to identify a nasal mucosa marker that allows the discrimination of nasal mucosa from saliva or vaginal mucosa and nosebleed blood from peripheral blood. An updated version of the multiplex was prepared to which the nasal mucosa marker was added and in which markers for semen, vaginal mucosa and blood were replaced. Lactobacillus markers were regarded unsuitable as replacement for vaginal mucosa mRNA markers because of background signals on penile swabs that appeared devoid of female DNA. Furthermore, we provide approaches to deal with highly unbalanced mixtures. First, a differential extraction protocol was incorporated into a co-extraction protocol to allow DNA and RNA analysis of separated non-sperm and sperm fractions. In a second approach, besides the standard multiplex, a customized multiplex is used which excludes markers for prevailing cell types. This allows the use of lower cDNA inputs for the prevailing cell types and higher inputs for cell types that appear masked. Additionally, we assessed the relation between the percentage of alleles or markers detected in DNA or RNA profiles when decreasing sample amounts are analysed. While blood, saliva, semen and menstrual secretion show the trend that DNA profiling is more sensitive than RNA profiling, the reverse is seen

  3. RNA interference-based therapeutics: molecular platforms for infectious diseases. (United States)

    Dyawanapelly, Sathish; Ghodke, Sharwari Bhagwat; Vishwanathan, Ramya; Dandekar, Prajakta; Jain, Ratnesh


    The potential uses and therapeutic benefits of RNA interference (RNAi) are enormous. Recent insights into RNAi technologies have highlighted their role in analyzing the functions and regulation of gene expression in eukaryotes and further utilizing this information for identification and amelioration of many diseases. These studies have also established the role of RNAi mediated post-transcriptional gene silencing (PTGS) mechanism in mammals by several endogenous, gene regulation systems including small interfering RNAs (siRNA), micro RNA (miRNA) and small hairpin RNAs (shRNA). Moreover, these RNAi-based therapeutics have demonstrated the capability to silence therapeutically relevant genes in various in vivo models of cancer, infections autoimmune diseases and other genetic disorders. Over the past few decades, infectious diseases have been one of the leading causes of death around the world. Ubiquitously, intracellular obligate or facultative microorganisms cause serious or fatal infections and associated diseases in humans. Currently available literature suggests that infections caused by intracellular pathogens present an intriguing area, wherein RNAi technology may be effectively employed to neutralize the harmful effects of various intracellular pathogens. In this manuscript, we have emphasized on the challenges and opportunities involved in the therapy of such intracellular infections, especially employing RNAi-based interventions. We have focused our discussion on the current state-of-the-art RNAi-based therapies, which have been explored for various intracellular infections mediated by bacteria, fungi, viruses and protozoa. Nanocarrier mediated delivery of siRNA and shRNA molecules have also been found to overcome the various delivery challenges of these biotherapeutics; these have also been briefly summarized here. Furthermore, the outcomes and progresses that have been made in pre-clinical models and clinical trials have also been presented to review the

  4. RNA-Based Assessment of Diversity and Composition of Active Archaeal Communities in the German Bight

    Directory of Open Access Journals (Sweden)

    Bernd Wemheuer


    Full Text Available Archaea play an important role in various biogeochemical cycles. They are known extremophiles inhabiting environments such as thermal springs or hydrothermal vents. Recent studies have revealed a significant abundance of Archaea in moderate environments, for example, temperate sea water. Nevertheless, the composition and ecosystem function of these marine archaeal communities is largely unknown. To assess diversity and composition of active archaeal communities in the German Bight, seven marine water samples were taken and studied by RNA-based analysis of ribosomal 16S rRNA. For this purpose, total RNA was extracted from the samples and converted to cDNA. Archaeal community structures were investigated by pyrosequencing-based analysis of 16S rRNA amplicons generated from cDNA. To our knowledge, this is the first study combining next-generation sequencing and metatranscriptomics to study archaeal communities in marine habitats. The pyrosequencing-derived dataset comprised 62,045 archaeal 16S rRNA sequences. We identified Halobacteria as the predominant archaeal group across all samples with increased abundance in algal blooms. Thermoplasmatales (Euryarchaeota and the Marine Group I (Thaumarchaeota were identified in minor abundances. It is indicated that archaeal community patterns were influenced by environmental conditions.

  5. Extraction of high-quality RNA from germinating barley (Hordeum vulgare L.) seeds containing high levels of starch. (United States)

    Comparative evaluation of gene expression levels can lead to improved understanding of the gene networks underlying traits of economic importance. Extraction of high-quality RNA from germinating barley seeds that contain high levels of starch is of vital importance for analysing the expression of ca...

  6. Evaluation of four different systems for extraction of RNA from stool suspensions using MS-2 coliphage as an exogenous control for RT-PCR inhibition.

    Directory of Open Access Journals (Sweden)

    Lester M Shulman

    Full Text Available Knowing when, and to what extent co-extracted inhibitors interfere with molecular RNA diagnostic assays is of utmost importance. The QIAamp Viral RNA Mini Kit (A; MagNA Pure LC2.0 Automatic extractor (B; KingFisher (C; and NucliSENS EasyMag (D RNA extraction systems were evaluated for extraction efficiency and co-purification of inhibitors from stool suspensions. Real-Time Reverse Transcriptase Polymerase Chain Reaction (rRT-PCR of MS-2 coliphage spiked into each system's lysis buffer served as an external control for both. Cycle thresholds (Cts of the MS2 were determined for RNA extracted from stool suspensions containing unknown (n = 93 or varying amounts of inhibitors (n = 92. Stool suspensions from the latter group were also used to determine whether MS-2 and enterovirus rRT-PCR inhibitions were correlated. Specifically 23 RNA extracts from stool suspensions were spiked with enterovirus RNA after extraction and 13 of these stool suspension were spiked with intact enterovirus before extraction. MS2 rRT-PCR inhibition varied for RNAs extracted by the different systems. Inhibition was noted in 12 (13.0%, 26 (28.3%, 7 (7.6%, and 7 (7.6% of the first 93 RNA extracts, and 58 (63.0%, 55 (59.8%, 37 (40.2% and 30 (32.6% of the second 92 extracts for A, B, C, and D, respectively. Furthermore, enterovirus rRT-PCR inhibition correlated with MS2 rRT-PCR inhibition for added enterovirus RNA or virus particles. In conclusion, rRT-PCR for MS-2 RNA is a good predictor of inhibition of enterovirus RNA extracted from stool suspensions. EasyMag performed the best, however all four extraction methods were suitable provided that external controls identified problematic samples.

  7. 辣椒叶片总RNA快速提取%Rapid Extraction of Total RNA from Capsicum annuum Leaves

    Institute of Scientific and Technical Information of China (English)

    李小霞; 肖仲久; 宋培勇; 周逊; 谢语


    Trizol extraction method was modified to extract total RNA from Capsicum annuum leaves. The result of agar gel electrophoresis, ultraviolet ray photometer and RT-PCR showed that the total RNA obtained by modified Trizol method was of high quality, and suitable for downstream applications.%采用改良的Trizol法对辣椒(Capsicum annuum)叶片的总RNA进行了提取,利用琼脂糖凝胶电泳、紫外分光光度法、RT-PCR进行RNA纯度、完整性检测.结果表明,Trizol法提取可获得较高质量的辣椒叶片总RNA,能满足后续的研究需要.

  8. Total RNA extraction of Cryptococcus neoformans%新生隐球菌总 RNA 抽提方法的实验研究

    Institute of Scientific and Technical Information of China (English)

    徐赤宇; 刘翠杰; 吴建华; 仇芸; 赵瑾; 朱红梅; 温海


    目的 探讨快速获取高质量的新生隐球菌总RNA的实验方法.方法 选取新生隐球菌的荚膜株、荚膜缺陷株,分别设计采用4种方法提取总RNA:酸洗玻璃珠法、液氮研磨法、异硫氰酸胍一步法、冷酸洗玻璃珠联合Yeast RNA kit法.用紫外线分光光度计测量其OD260、0D280的值,并且进行琼脂糖凝胶电泳,同时应用定量PCR法鉴定RNA质量.结果 酸洗玻璃珠法、液氮研磨法、异硫氰酸胍一步法、冷酸洗玻璃珠联合Yeast RNA kit法的RNA产量分别为0.2μg/105细胞、0.4.μg/105细胞、0.1 μg/105细胞、0.6 μg/105细胞.结论 冷酸洗玻璃珠联合Yeast RNA kit法提取的RNA均一性和完整性最好,是简便、快捷地提取具有荚膜和细胞壁双重屏障的新生隐球菌RNA的理想方法.%Objective A quick approach to extract high quality total RNA from Cryptococcus neoformans plays a vital role in researches on the molecular detection and disease-related gene expression of high pathogenic Cryptococcus neoformans . Method Cryptoccocus neoformans strain and Cryptoccocus neoformans capsule deficient strain were used in the research. Four methods were designed to perform the extraction: acid washed glass beads approach, liquid nitrogen approach, guanidinium isothiocyanate one step approach and acid washed glass beads associated with Yeast RNA kit approach. RNA quality is determined by measurement of OD260 and OD280 with UV spectrophotometer, by agarose gel electrophoresis and by quantitative PCR. Result RNA yield of the four approaches, namely, acid washed glass beads approach, liquid nitrogen approach, guanidinium isothiocyanate one step approach and acid washed glass beads associated with Yeast RNA kit approach, was 0. 2 μg/10 cells, 0.4 μg/10 cells, 0. 1 μg/10 cells and 0. 6 μg/105 cells respectively. Conclusion RNA which gained by the approach of acid washed glass beads associated with Yeast RNA kit has the best homogeneity and integrity. Thus, this


    Directory of Open Access Journals (Sweden)

    R. Aishwarya


    Full Text Available The control of prosthetic limb would be more effective if it is based on Surface Electromyogram (SEMG signals from remnant muscles. The analysis of SEMG signals depend on a number of factors, such as amplitude as well as time- and frequency-domain properties. Time series analysis using Auto Regressive (AR model and Mean frequency which is tolerant to white Gaussian noise are used as feature extraction techniques. EMG Histogram is used as another feature vector that was seen to give more distinct classification. The work was done with SEMG dataset obtained from the NINAPRO DATABASE, a resource for bio robotics community. Eight classes of hand movements hand open, hand close, Wrist extension, Wrist flexion, Pointing index, Ulnar deviation, Thumbs up, Thumb opposite to little finger are taken into consideration and feature vectors are extracted. The feature vectors can be given to an artificial neural network for further classification in controlling the prosthetic arm which is not dealt in this paper.

  10. Short Communication An efficient method for simultaneous extraction of high-quality RNA and DNA from various plant tissues. (United States)

    Oliveira, R R; Viana, A J C; Reátegui, A C E; Vincentz, M G A


    Determination of gene expression is an important tool to study biological processes and relies on the quality of the extracted RNA. Changes in gene expression profiles may be directly related to mutations in regulatory DNA sequences or alterations in DNA cytosine methylation, which is an epigenetic mark. Correlation of gene expression with DNA sequence or epigenetic mark polymorphism is often desirable; for this, a robust protocol to isolate high-quality RNA and DNA simultaneously from the same sample is required. Although commercial kits and protocols are available, they are mainly optimized for animal tissues and, in general, restricted to RNA or DNA extraction, not both. In the present study, we describe an efficient and accessible method to extract both RNA and DNA simultaneously from the same sample of various plant tissues, using small amounts of starting material. The protocol was efficient in the extraction of high-quality nucleic acids from several Arabidopsis thaliana tissues (e.g., leaf, inflorescence stem, flower, fruit, cotyledon, seedlings, root, and embryo) and from other tissues of non-model plants, such as Avicennia schaueriana (Acanthaceae), Theobroma cacao (Malvaceae), Paspalum notatum (Poaceae), and Sorghum bicolor (Poaceae). The obtained nucleic acids were used as templates for downstream analyses, such as mRNA sequencing, quantitative real time-polymerase chain reaction, bisulfite treatment, and others; the results were comparable to those obtained with commercial kits. We believe that this protocol could be applied to a broad range of plant species, help avoid technical and sampling biases, and facilitate several RNA- and DNA-dependent analyses.

  11. (Nontranslational medicine: RNA-based therapeutics in bacteria

    Directory of Open Access Journals (Sweden)

    Adam M Dinan


    Full Text Available The rise and spread of antibiotic resistance is among the most severe challenges facing modern medicine. Despite this fact, attempts to develop novel classes of antibiotic have been largely unsuccessful. The traditional mechanisms by which antibiotics work are subject to relatively rapid bacterial resistance via mutation, and hence have a limited period of efficacy. One promising strategy to ameliorate this problem is to shift from the use of chemical compounds targeting protein structures and processes to a new era of RNA-based therapeutics. RNA-mediated regulation (riboregulation has evolved naturally in bacteria and is therefore a highly efficient means by which gene expression can be manipulated. Here, we describe recent advances towards the development of effective anti-bacterial therapies, which operate through various strategies centred on RNA. Significant challenges facing the field, including the identification of suitable molecular targets and delivery strategies, will also be considered.

  12. Web Template Extraction Based on Hyperlink Analysis

    Directory of Open Access Journals (Sweden)

    Julián Alarte


    Full Text Available Web templates are one of the main development resources for website engineers. Templates allow them to increase productivity by plugin content into already formatted and prepared pagelets. For the final user templates are also useful, because they provide uniformity and a common look and feel for all webpages. However, from the point of view of crawlers and indexers, templates are an important problem, because templates usually contain irrelevant information such as advertisements, menus, and banners. Processing and storing this information is likely to lead to a waste of resources (storage space, bandwidth, etc.. It has been measured that templates represent between 40% and 50% of data on the Web. Therefore, identifying templates is essential for indexing tasks. In this work we propose a novel method for automatic template extraction that is based on similarity analysis between the DOM trees of a collection of webpages that are detected using menus information. Our implementation and experiments demonstrate the usefulness of the technique.

  13. Identification of miRNA-mRNA Modules in Colorectal Cancer Using Rough Hypercuboid Based Supervised Clustering (United States)

    Paul, Sushmita; Lakatos, Petra; Hartmann, Arndt; Schneider-Stock, Regine; Vera, Julio


    Differences in the expression profiles of miRNAs and mRNAs have been reported in colorectal cancer. Nevertheless, information on important miRNA-mRNA regulatory modules in colorectal cancer is still lacking. In this regard, this study presents an application of the RH-SAC algorithm on miRNA and mRNA expression data for identification of potential miRNA-mRNA modules. First, a set of miRNA rules was generated using the RH-SAC algorithm. The mRNA targets of the selected miRNAs were identified using the miRTarBase database. Next, the expression values of target mRNAs were used to generate mRNA rules using the RH-SAC. Then all miRNA-mRNA rules have been integrated for generating networks. The RH-SAC algorithm unlike other existing methods selects a group of co-expressed miRNAs and mRNAs that are also differentially expressed. In total 17 miRNAs and 141 mRNAs were selected. The enrichment analysis of selected mRNAs revealed that our method selected mRNAs that are significantly associated with colorectal cancer. We identified novel miRNA/mRNA interactions in colorectal cancer. Through experiment, we could confirm that one of our discovered miRNAs, hsa-miR-93-5p, was significantly up-regulated in 75.8% CRC in comparison to their corresponding non-tumor samples. It could have the potential to examine colorectal cancer subtype specific unique miRNA/mRNA interactions. PMID:28220871

  14. Comparison of RNA extraction kits and histological stains for laser capture microdissected prostate tissue

    NARCIS (Netherlands)

    K. Kolijn (Kimberley); G.J.H.L. Leenders (Geert)


    textabstractBackground Laser capture microdissection offers unique possibilities for the isolation of specific cell populations or histological structures. However, isolation of RNA from microdissected tissue is challenging due to degradation and minimal yield of RNA during laser capture microdisse

  15. Extractions of High Quality RNA from the Seeds of Jerusalem Artichoke and Other Plant Species with High Levels of Starch and Lipid

    Directory of Open Access Journals (Sweden)

    Tanupat Mornkham


    Full Text Available Jerusalem artichoke (Helianthus tuberosus L. is an important tuber crop. However, Jerusalem artichoke seeds contain high levels of starch and lipid, making the extraction of high-quality RNA extremely difficult and the gene expression analysis challenging. This study was aimed to improve existing methods for extracting total RNA from Jerusalem artichoke dry seeds and to assess the applicability of the improved method in other plant species. Five RNA extraction methods were evaluated on Jerusalem artichoke seeds and two were modified. One modified method with the significant improvement was applied to assay seeds of diverse Jerusalem artichoke accessions, sunflower, rice, maize, peanut and marigold. The effectiveness of the improved method to extract total RNA from seeds was assessed using qPCR analysis of four selected genes. The improved method of Ma and Yang (2011 yielded a maximum RNA solubility and removed most interfering substances. The improved protocol generated 29 to 41 µg RNA/30 mg fresh weight. An A260/A280 ratio of 1.79 to 2.22 showed their RNA purity. Extracted RNA was effective for downstream applications such as first-stranded cDNA synthesis, cDNA cloning and qPCR. The improved method was also effective to extract total RNA from seeds of sunflower, rice, maize and peanut that are rich in polyphenols, lipids and polysaccharides.

  16. Fingerprint Feature Extraction Based on Macroscopic Curvature

    Institute of Scientific and Technical Information of China (English)

    Zhang Xiong; He Gui-ming; Zhang Yun


    In the Automatic Fingerprint Identification System (AFIS), extracting the feature of fingerprint is very important. The local curvature of ridges of fingerprint is irregular, so people have the barrier to effectively extract the fingerprint curve features to describe fingerprint. This article proposes a novel algorithm; it embraces information of few nearby fingerprint ridges to extract a new characteristic which can describe the curvature feature of fingerprint. Experimental results show the algorithm is feasible, and the characteristics extracted by it can clearly show the inner macroscopic curve properties of fingerprint. The result also shows that this kind of characteristic is robust to noise and pollution.

  17. Fingerprint Feature Extraction Based on Macroscopic Curvature

    Institute of Scientific and Technical Information of China (English)

    Zhang; Xiong; He; Gui-Ming; 等


    In the Automatic Fingerprint Identification System(AFIS), extracting the feature of fingerprint is very important. The local curvature of ridges of fingerprint is irregular, so people have the barrier to effectively extract the fingerprint curve features to describe fingerprint. This article proposes a novel algorithm; it embraces information of few nearby fingerprint ridges to extract a new characterstic which can describe the curvature feature of fingerprint. Experimental results show the algorithm is feasible, and the characteristics extracted by it can clearly show the inner macroscopic curve properties of fingerprint. The result also shows that this kind of characteristic is robust to noise and pollution.

  18. A multiplex endpoint RT-PCR assay for quality assessment of RNA extracted from formalin-fixed paraffin-embedded tissues

    Directory of Open Access Journals (Sweden)

    Dobrovic Alexander


    Full Text Available Abstract Background RNA extracted from formalin-fixed paraffin-embedded (FFPE samples is chemically modified and degraded, which compromises its use in gene expression studies. Most of the current approaches for RNA quality assessment are not suitable for FFPE derived RNA. Results We have developed a single-tube multiplex endpoint RT-PCR assay specifically designed to evaluate RNA extracted from FFPE tissues for mRNA integrity and performance in reverse transcription - quantitative real-time PCR (RT-qPCR assays. This single-tube quality control (QC assay minimises the amount of RNA used in quality control. mRNA integrity and the suitability of RNA for RT-PCR is evaluated by the multiplex endpoint RT-PCR assay using the TBP gene mRNA as the target sequence. The RT-PCR amplicon sizes, 92, 161, 252 and 300 bp, cover a range of amplicon sizes suitable for a wide range of RT-qPCR assays. The QC assay was used to evaluate RNA prepared by two different protocols for extracting total RNA from needle microdissected FFPE breast tumour samples. The amplification products were analysed by gel electrophoresis where the spectrum of amplicon sizes indicated the level of RNA degradation and thus the suitability of the RNA for PCR. The ability of the multiplex endpoint RT-PCR QC assay to identify FFPE samples with an adequate RNA quality was validated by examining the Cq values of an RT-qPCR assay with an 87 bp amplicon. Conclusions The multiplex endpoint RT-PCR assay is well suited for the determination of the quality of FFPE derived RNAs, to identify which RT-PCR assays they are suitable for, and is also applicable to assess non-FFPE RNA for gene expression studies. Furthermore, the assay can also be used for the evaluation of RNA extraction protocols from FFPE samples.

  19. Shape based indexing for faster search of RNA family databases

    Directory of Open Access Journals (Sweden)

    Reeder Jens


    Full Text Available Abstract Background Most non-coding RNA families exert their function by means of a conserved, common secondary structure. The Rfam data base contains more than five hundred structurally annotated RNA families. Unfortunately, searching for new family members using covariance models (CMs is very time consuming. Filtering approaches that use the sequence conservation to reduce the number of CM searches, are fast, but it is unknown to which sacrifice. Results We present a new filtering approach, which exploits the family specific secondary structure and significantly reduces the number of CM searches. The filter eliminates approximately 85% of the queries and discards only 2.6% true positives when evaluating Rfam against itself. First results also capture previously undetected non-coding RNAs in a recent human RNAz screen. Conclusion The RNA shape index filter (RNAsifter is based on the following rationale: An RNA family is characterised by structure, much more succinctly than by sequence content. Structures of individual family members, which naturally have different length and sequence composition, may exhibit structural variation in detail, but overall, they have a common shape in a more abstract sense. Given a fixed release of the Rfam data base, we can compute these abstract shapes for all families. This is called a shape index. If a query sequence belongs to a certain family, it must be able to fold into the family shape with reasonable free energy. Therefore, rather than matching the query against all families in the data base, we can first (and quickly compute its feasible shape(s, and use the shape index to access only those families where a good match is possible due to a common shape with the query.

  20. Idioms-based Business Rule Extraction

    NARCIS (Netherlands)

    Smit, R


    This thesis studies the extraction of embedded business rules, using the idioms of the used framework to identify them. Embedded business rules exist as source code in the software system and knowledge about them may get lost. Extraction of those business rules could make them accessible and managea

  1. Co-LncRNA: investigating the lncRNA combinatorial effects in GO annotations and KEGG pathways based on human RNA-Seq data. (United States)

    Zhao, Zheng; Bai, Jing; Wu, Aiwei; Wang, Yuan; Zhang, Jinwen; Wang, Zishan; Li, Yongsheng; Xu, Juan; Li, Xia


    Long non-coding RNAs (lncRNAs) are emerging as key regulators of diverse biological processes and diseases. However, the combinatorial effects of these molecules in a specific biological function are poorly understood. Identifying co-expressed protein-coding genes of lncRNAs would provide ample insight into lncRNA functions. To facilitate such an effort, we have developed Co-LncRNA, which is a web-based computational tool that allows users to identify GO annotations and KEGG pathways that may be affected by co-expressed protein-coding genes of a single or multiple lncRNAs. LncRNA co-expressed protein-coding genes were first identified in publicly available human RNA-Seq datasets, including 241 datasets across 6560 total individuals representing 28 tissue types/cell lines. Then, the lncRNA combinatorial effects in a given GO annotations or KEGG pathways are taken into account by the simultaneous analysis of multiple lncRNAs in user-selected individual or multiple datasets, which is realized by enrichment analysis. In addition, this software provides a graphical overview of pathways that are modulated by lncRNAs, as well as a specific tool to display the relevant networks between lncRNAs and their co-expressed protein-coding genes. Co-LncRNA also supports users in uploading their own lncRNA and protein-coding gene expression profiles to investigate the lncRNA combinatorial effects. It will be continuously updated with more human RNA-Seq datasets on an annual basis. Taken together, Co-LncRNA provides a web-based application for investigating lncRNA combinatorial effects, which could shed light on their biological roles and could be a valuable resource for this community. Database URL:

  2. Long dsRNA-mediated RNA interference and immunostimulation: a targeted delivery approach using polyethyleneimine based nano-carriers. (United States)

    Sajeesh, S; Lee, Tae Yeon; Hong, Sun Woo; Dua, Pooja; Choe, Jeong Yong; Kang, Aeyeon; Yun, Wan Soo; Song, Changsik; Park, Sung Ha; Kim, Soyoun; Li, Chiang; Lee, Dong-Ki


    RNA oligonucleotides capable of inducing controlled immunostimulation combined with specific oncogene silencing via an RNA interference (RNAi) mechanism provide synergistic inhibition of cancer cell growth. With this concept, we previously designed a potent immunostimulatory long double stranded RNA, referred to as liRNA, capable of executing RNAi mediated specific target gene silencing. In this study, we developed a highly effective liRNA based targeted delivery system to apply in the treatment of glioblastoma multiforme. A stable nanocomplex was fabricated by complexing multimerized liRNA structures with cross-linked branched poly(ethylene imine) (bPEI) via electrostatic interactions. We show clear evidence that the cross-linked bPEI was quite effective in enhancing the cellular uptake of liRNA on U87MG cells. Moreover, the liRNA-PEI nanocomplex provided strong RNAi mediated target gene silencing compared to that of the conventional siRNA-PEI complex. Further, the bPEI modification strategy with specific ligand attachment assisted the uptake of the liRNA-PEI complex on the mouse brain endothelial cell line (b.End3). Such delivery systems combining the beneficial elements of targeted delivery, controlled immunostimulation, and RNAi mediated target silencing have immense potential in anticancer therapy.

  3. An optmized protocol for simultaneous extraction of DNA and RNA from soils Protocolo otimizado para extração simultanea de DNA e RNA de solo

    Directory of Open Access Journals (Sweden)

    Rodrigo Costa


    Full Text Available In this work we report an optimized protocol for simultaneous extraction of DNA and RNA from soil matrices. Treatment of soil matrices with ethanol followed by bead-beating worked as a successful strategy to lyse the cells without considerable degradation of nucleic acids, resulting in DNA and RNA of good yield and integrity. The reverse transcribed RNA could be amplified with primers targeting a glutamine synthetase (glnA gene fragment. From both DNA and cDNA, 16S rDNA fragments were amplified and analyzed by Denaturing Gradient Gel Electrophoresis (DGGE. The method was applied to soil and rhizosphere (strawberry and oilseed rape samples. Two other protocols for the extraction of nucleic acids from soil were applied to the same set of samples in order to compare the methods in terms of efficiency and reproducibility. The DGGE profiles indicated no relevant differences between the patterns obtained. The method described here is suitable for rapid processing of many samples and therefore appropriate for ecological studies.Nesse trabalho descrevemos um protocolo otimizado para extração simultânea de DNA e RNA de solo. O tratamento das amostras de solo com etanol e posterior agitação com partículas foi uma estratégia bem sucedida para lise das células sem degradação significativa dos ácidos nucléicos, resultando em bom rendimento de DNA e RNA íntegros. O RNA transcrito pode ser amplificado com iniciadores com alvo no fragmento do gene da glutamina sintetase (glnA. Os fragmentos 16S rDNA, tanto do DNA como do cDNA, foram amplificados e analisados por DGGE. O método foi aplicado para amostras de solo e rizosfera (morango e canola. Dois outros protocolos para extração de ácidos nucléicos de solo foram aplicados para o mesmo lote de amostras, de forma a comparar os métodos quanto à eficiência e reprodutibilidade. Os perfis de DGGE mostraram não haver diferença relevante nos padrões obtidos. O método descrito é apropriado para

  4. A unique method for isolation and solubilization of proteins after extraction of RNA from tumor tissue using trizol. (United States)

    Likhite, Neah; Warawdekar, Ujjwala M


    The aim of this study was to develop a systems approach to study tumor tissue. The importance of concurrent extraction of RNA, DNA, and protein is evident when genetic aberrations and the differences in the proteome and transcriptome have to be correlated. The need is magnified, as the tissue available for study is miniscule, is shared amongst investigators, and needs to support the holistic approach. Trizol is a monophasic solution of phenol and guanidine isothiocyanate and can be used to isolate the three biomolecules simultaneously. Trizol solution was used for RNA extraction in an ongoing study about expression of molecular markers in non-small cell lung carcinoma (NSCLC) and breast tumor tissue. After isolation of RNA, the remaining Trizol fraction was stored at -80°C for over 6 months. We have shown the extraction of protein from 17 tumor and adjacent, normal tissue samples and PBMC obtained from four blood samples. The isolation and solubilization of the protein fraction were done according to the product information using isopropanol for precipitation and guanidine hydrochloride and SDS for washing and solubilization, respectively, modifying the time of solubilization. The protein was estimated by the bicinchoninic acid (BCA) method and analyzed on polyacrylamide gels. Staining showed a wide repertoire, and Western blotting confirmed extraction of cytokeratins (CK) and DNA repair proteins. Whereas tissue samples in which the RNA was degraded could be assessed by the presence of the protein salvaging the marker analysis, it was seen that nuclear proteins cannot be retrieved and are probably lost with the DNA fraction.

  5. Transparent DNA/RNA Co-extraction Workflow Protocol Suitable for Inhibitor-Rich Environmental Samples That Focuses on Complete DNA Removal for Transcriptomic Analyses. (United States)

    Lim, Natalie Y N; Roco, Constance A; Frostegård, Åsa


    Adequate comparisons of DNA and cDNA libraries from complex environments require methods for co-extraction of DNA and RNA due to the inherent heterogeneity of such samples, or risk bias caused by variations in lysis and extraction efficiencies. Still, there are few methods and kits allowing simultaneous extraction of DNA and RNA from the same sample, and the existing ones generally require optimization. The proprietary nature of kit components, however, makes modifications of individual steps in the manufacturer's recommended procedure difficult. Surprisingly, enzymatic treatments are often performed before purification procedures are complete, which we have identified here as a major problem when seeking efficient genomic DNA removal from RNA extracts. Here, we tested several DNA/RNA co-extraction commercial kits on inhibitor-rich soils, and compared them to a commonly used phenol-chloroform co-extraction method. Since none of the kits/methods co-extracted high-quality nucleic acid material, we optimized the extraction workflow by introducing small but important improvements. In particular, we illustrate the need for extensive purification prior to all enzymatic procedures, with special focus on the DNase digestion step in RNA extraction. These adjustments led to the removal of enzymatic inhibition in RNA extracts and made it possible to reduce genomic DNA to below detectable levels as determined by quantitative PCR. Notably, we confirmed that DNase digestion may not be uniform in replicate extraction reactions, thus the analysis of "representative samples" is insufficient. The modular nature of our workflow protocol allows optimization of individual steps. It also increases focus on additional purification procedures prior to enzymatic processes, in particular DNases, yielding genomic DNA-free RNA extracts suitable for metatranscriptomic analysis.

  6. Prediction of RNA Secondary Structure Based on Particle Swarm Optimization

    Institute of Scientific and Technical Information of China (English)

    LIU Yuan-ning; DONG Hao; ZHANG Hao; WANG Gang; LI Zhi; CHEN Hui-ling


    A novel method for the prediction of RNA secondary structure was proposed based on the particle swarm optimization(PSO). PSO is known to be effective in solving many different types of optimization problems and known for being able to approximate the global optimal results in the solution space. We designed an efficient objective function according to the minimum free energy, the number of selected stems and the average length of selected stems. We calculated how many legal stems there were in the sequence, and selected some of them to obtain an optimal result using PSO in the right of the objective function. A method based on the improved particle swarm optimization(IPSO) was proposed to predict RNA secondary structure, which consisted of three stages. The first stage was applied to e ncoding the source sequences, and to exploring all the legal stems. Then, a set of encoded stems were created in order to prepare input data for the second stage. In the second stage, IPSO was responsible for structure selection. At last, the optimal result was obtained from the secondary structures selected via IPSO. Nine sequences from the comparative RNA website were selected for the evaluation of the proposed method. Compared with other six methods, the proposed method decreased the complexity and enhanced the sensitivity and specificity on the basis of the experiment results.

  7. Single-Step RNA Extraction from Different Hydrogel-Embedded Mesenchymal Stem Cells for Quantitative Reverse Transcription-Polymerase Chain Reaction Analysis. (United States)

    Köster, Natascha; Schmiermund, Alexandra; Grubelnig, Stefan; Leber, Jasmin; Ehlicke, Franziska; Czermak, Peter; Salzig, Denise


    For many tissue engineering applications, cells such as human mesenchymal stem cells (hMSCs) must be embedded in hydrogels. The analysis of embedded hMSCs requires RNA extraction, but common extraction procedures often produce low yields and/or poor quality RNA. We systematically investigated four homogenization methods combined with eight RNA extraction protocols for hMSCs embedded in three common hydrogel types (alginate, agarose, and gelatin). We found for all three hydrogel types that using liquid nitrogen or a rotor-stator produced low RNA yields, whereas using a microhomogenizer or enzymatic/chemical hydrogel digestion achieved better yields regardless of which extraction protocol was subsequently applied. The hot phenol extraction protocol generally achieved the highest A260 values (representing up to 40.8 μg RNA per 10(6) cells), but the cetyltrimethylammonium bromide (CTAB) method produced RNA of better quality, with A260/A280 and A260/A230 ratios and UV spectra similar to the pure RNA control. The RNA produced by this method was also suitable as a template for endpoint and quantitative reverse transcription-PCR (qRT-PCR), achieving low Ct values of ∼20. The prudent choice of hydrogel homogenization and RNA extraction methods can ensure the preparation of high-quality RNA that generates reliable endpoint and quantitative RT-PCR data. We therefore propose a universal method that is suitable for the extraction of RNA from cells embedded in all three hydrogel types commonly used for tissue engineering.

  8. A universal BMV-based RNA recombination system--how to search for general rules in RNA recombination. (United States)

    Alejska, Magdalena; Figlerowicz, Magdalena; Malinowska, Nelli; Urbanowicz, Anna; Figlerowicz, Marek


    At present, there is no doubt that RNA recombination is one of the major factors responsible for the generation of new RNA viruses and retroviruses. Numerous experimental systems have been created to investigate this complex phenomenon. Consequently, specific RNA structural motifs mediating recombination have been identified in several viruses. Unfortunately, up till now a unified model of genetic RNA recombination has not been formulated, mainly due to difficulties with the direct comparison of data obtained for different RNA-based viruses. To solve this problem, we have attempted to construct a universal system in which the recombination activity of various RNA sequences could be tested. To this end, we have used brome mosaic virus, a model (+)RNA virus of plants, for which the structural requirements of RNA recombination are well defined. The effectiveness of the new homomolecular system has been proven in an experiment involving two RNA sequences derived from the hepatitis C virus genome. In addition, comparison of the data obtained with the homomolecular system with those generated earlier using the heteromolecular one has provided new evidence that the mechanisms of homologous and non-homologous recombination are different and depend on the virus' mode of replication.

  9. Optimization of the RNA extraction method for transcriptome studies of Salmonella inoculated on commercial raw chicken breast samples

    Directory of Open Access Journals (Sweden)

    Muthaiyan Arunachalam


    Full Text Available Abstract Background There has been increased interest in the study of molecular survival mechanisms expressed by foodborne pathogens present on food surfaces. Determining genomic responses of these pathogens to antimicrobials is of particular interest since this helps to understand antimicrobial effects at the molecular level. Assessment of bacterial gene expression by transcriptomic analysis in response to these antimicrobials would aid prediction of the phenotypic behavior of the bacteria in the presence of antimicrobials. However, before transcriptional profiling approaches can be implemented routinely, it is important to develop an optimal method to consistently recover pathogens from the food surface and ensure optimal quality RNA so that the corresponding gene expression analysis represents the current response of the organism. Another consideration is to confirm that there is no interference from the "background" food or meat matrix that could mask the bacterial response. Findings Our study involved developing a food model system using chicken breast meat inoculated with mid-log Salmonella cells. First, we tested the optimum number of Salmonella cells required on the poultry meat in order to extract high quality RNA. This was analyzed by inoculating 10-fold dilutions of Salmonella on the chicken samples followed by RNA extraction. Secondly, we tested the effect of two different bacterial cell recovery solutions namely 0.1% peptone water and RNAprotect (Qiagen Inc. on the RNA yield and purity. In addition, we compared the efficiency of sonication and bead beater methods to break the cells for RNA extraction. To check chicken nucleic acid interference on downstream Salmonella microarray experiments both chicken and Salmonella cDNA labeled with different fluorescent dyes were mixed together and hybridized on a single Salmonella array. Results of this experiment did not show any cross-hybridization signal from the chicken nucleic acids. In

  10. [Characterization of Black and Dichothrix Cyanobacteria Based on the 16S Ribosomal RNA Gene Sequence (United States)

    Ortega, Maya


    My project focuses on characterizing different cyanobacteria in thrombolitic mats found on the island of Highborn Cay, Bahamas. Thrombolites are interesting ecosystems because of the ability of bacteria in these mats to remove carbon dioxide from the atmosphere and mineralize it as calcium carbonate. In the future they may be used as models to develop carbon sequestration technologies, which could be used as part of regenerative life systems in space. These thrombolitic communities are also significant because of their similarities to early communities of life on Earth. I targeted two cyanobacteria in my research, Dichothrix spp. and whatever black is, since they are believed to be important to carbon sequestration in these thrombolitic mats. The goal of my summer research project was to molecularly identify these two cyanobacteria. DNA was isolated from each organism through mat dissections and DNA extractions. I ran Polymerase Chain Reactions (PCR) to amplify the 16S ribosomal RNA (rRNA) gene in each cyanobacteria. This specific gene is found in almost all bacteria and is highly conserved, meaning any changes in the sequence are most likely due to evolution. As a result, the 16S rRNA gene can be used for bacterial identification of different species based on the sequence of their 16S rRNA gene. Since the exact sequence of the Dichothrix gene was unknown, I designed different primers that flanked the gene based on the known sequences from other taxonomically similar cyanobacteria. Once the 16S rRNA gene was amplified, I cloned the gene into specialized Escherichia coli cells and sent the gene products for sequencing. Once the sequence is obtained, it will be added to a genetic database for future reference to and classification of other Dichothrix sp.

  11. Quantitative and Sensitive RNA Based Detection of Bacillus Spores

    Directory of Open Access Journals (Sweden)

    Ekaterina eOsmekhina


    Full Text Available The fast and reliable detection of bacterial spores is of great importance and still remains a challenge. Here we describe a direct RNA based diagnostic method for the specific detection of viable bacterial spores which does not depends on an enzymatic amplification step and therefore is directly appropriate for quantification. The procedure includes the following steps: (i heat activation of spores, (ii germination and enrichment cultivation, (iii cell lysis, and (iv analysis of 16S rRNA in crude cell lysates using a sandwich hybridization assay. The sensitivity of the method is dependent on the cultivation time and the detection limit; it is possible to detect 10 spores per ml when the RNA analysis is performed after 6 h of enrichment cultivation. At spore concentrations above 106 spores per ml the cultivation time can be shortened to 30 min. Total analysis times are in the range of 2 to 8 hours depending on the spore concentration in samples. The developed procedure is optimized at the example of Bacillus subtilis spores but should be applicable to other organisms. The new method can easily be modified for other target RNAs and is suitable for specific detection of spores from known groups of organisms.

  12. RNA 3D Structure Modeling by Combination of Template-Based Method ModeRNA, Template-Free Folding with SimRNA, and Refinement with QRNAS. (United States)

    Piatkowski, Pawel; Kasprzak, Joanna M; Kumar, Deepak; Magnus, Marcin; Chojnowski, Grzegorz; Bujnicki, Janusz M


    RNA encompasses an essential part of all known forms of life. The functions of many RNA molecules are dependent on their ability to form complex three-dimensional (3D) structures. However, experimental determination of RNA 3D structures is laborious and challenging, and therefore, the majority of known RNAs remain structurally uncharacterized. To address this problem, computational structure prediction methods were developed that either utilize information derived from known structures of other RNA molecules (by way of template-based modeling) or attempt to simulate the physical process of RNA structure formation (by way of template-free modeling). All computational methods suffer from various limitations that make theoretical models less reliable than high-resolution experimentally determined structures. This chapter provides a protocol for computational modeling of RNA 3D structure that overcomes major limitations by combining two complementary approaches: template-based modeling that is capable of predicting global architectures based on similarity to other molecules but often fails to predict local unique features, and template-free modeling that can predict the local folding, but is limited to modeling the structure of relatively small molecules. Here, we combine the use of a template-based method ModeRNA with a template-free method SimRNA. ModeRNA requires a sequence alignment of the target RNA sequence to be modeled with a template of the known structure; it generates a model that predicts the structure of a conserved core and provides a starting point for modeling of variable regions. SimRNA can be used to fold small RNAs (models for larger RNAs that have a correctly modeled core. ModeRNA can be either downloaded, compiled and run locally or run through a web interface at . SimRNA is currently available to download for local use as a precompiled software package at and as a

  13. Object Extraction Based on Evolutionary Morphological Processing

    Institute of Scientific and Technical Information of China (English)

    LI Bin; PAN Li


    This paper introduces a novel technique for object detection using genetic algorithms and morphological processing. The method employs a kind of object oriented structure element, which is derived by genetic algorithms. The population of morphological filters is iteratively evaluated according to a statistical performance index corresponding to object extraction ability, and evolves into an optimal structuring element using the evolution principles of genetic search. Experimental results of road extraction from high resolution satellite images are presented to illustrate the merit and feasibility of the proposed method.

  14. Assessing the Fecal Microbiota: An Optimized Ion Torrent 16S rRNA Gene-Based Analysis Protocol (United States)

    Foroni, Elena; Duranti, Sabrina; Turroni, Francesca; Lugli, Gabriele Andrea; Sanchez, Borja; Martín, Rebeca; Gueimonde, Miguel; van Sinderen, Douwe; Margolles, Abelardo; Ventura, Marco


    Assessing the distribution of 16S rRNA gene sequences within a biological sample represents the current state-of-the-art for determination of human gut microbiota composition. Advances in dissecting the microbial biodiversity of this ecosystem have very much been dependent on the development of novel high-throughput DNA sequencing technologies, like the Ion Torrent. However, the precise representation of this bacterial community may be affected by the protocols used for DNA extraction as well as by the PCR primers employed in the amplification reaction. Here, we describe an optimized protocol for 16S rRNA gene-based profiling of the fecal microbiota. PMID:23869230

  15. Assessing the fecal microbiota: an optimized ion torrent 16S rRNA gene-based analysis protocol.

    Directory of Open Access Journals (Sweden)

    Christian Milani

    Full Text Available Assessing the distribution of 16S rRNA gene sequences within a biological sample represents the current state-of-the-art for determination of human gut microbiota composition. Advances in dissecting the microbial biodiversity of this ecosystem have very much been dependent on the development of novel high-throughput DNA sequencing technologies, like the Ion Torrent. However, the precise representation of this bacterial community may be affected by the protocols used for DNA extraction as well as by the PCR primers employed in the amplification reaction. Here, we describe an optimized protocol for 16S rRNA gene-based profiling of the fecal microbiota.

  16. Gene expression profiling of RNA extracted from FFPE tissues: NuGEN technologies' whole-transcriptome amplification system. (United States)

    Turner, Leah; Heath, Joe Don; Kurn, Nurith


    Gene expression profiling of RNA isolated from formalin fixed, paraffin-embedded (FFPE) tissue samples has been historically challenging. Yet FFPE samples are sought-after because of the in-depth retrospective records typically associated with them rendering these samples a valuable resource for translational medicine studies. Extensive degradation, chemical modifications, and cross-linking have made it difficult to isolate RNA of sufficient quality required for large-scale gene expression profiling studies. NuGEN Technologies' WT-Ovation™ FFPE System linearly amplifies RNA from FFPE samples through a robust and simple whole-transcriptome approach using as little as 50 ng total RNA isolated from FFPE samples. The amplified material may be labeled with validated kits and/or protocols from NuGEN for analysis on any of the major gene expression microarray platforms, including: Affymetrix, Agilent, and Illumina gene expression arrays. Results compare well with those obtained using RNA from fresh-frozen samples. RNA quality from FFPE samples varies significantly and neither sample age nor sample size analysis via gel electrophoresis or the Agilent Bioanalyzer system accurately predict materials suitable for amplification. Therefore, NuGEN has validated a correlative qPCR-based analytical method for the RNA derived from FFPE samples which effectively predicts array results. The NuGEN approach enables fast and successful analysis of samples previously thought to be too degraded for gene expression analysis.

  17. A Novel Microfluidic Device for Fully Automated Extraction of RNA from Cell Cultures Project (United States)

    National Aeronautics and Space Administration — Differential gene expression by RNA profiling is a universal and critical step in space biology experiments, which seek to link specific molecular events with...

  18. A Novel Microfluidic Device for Fully Automated Extraction of RNA from Cell Cultures Project (United States)

    National Aeronautics and Space Administration — Obtaining high quality, intact RNA from cells is an ubiquitous need in the pursuit of space biology. Our overall objective is to develop and commercialize a...

  19. RNA extraction from various recalcitrant plant tissues with a cethyltrimethylammonium bromide-containing buffer followed by an acid guanidium thiocyanate-phenol-chloroform treatment. (United States)

    Suzuki, Yuji; Mae, Tadahiko; Makino, Amane


    High-quality total RNA was extracted using a cethyltrimethylammonium bromide-containing buffer followed by an acid guanidium thiocyanate-phenol-chloroform treatment from recalcitrant plant tissues such as tree leaves (pine, Norway spruce, ginkgo, Japanese cedar, rose), flowers (rose, Lotus japonicus) and storage tissues (seeds of Lotus japonicus and rice, sweet potato tuber, banana fruit). This protocol greatly reduced the time required for RNA extraction.

  20. Crude extracts of bacterially expressed dsRNA can be used to protect plants against virus infections

    Directory of Open Access Journals (Sweden)

    Vargas Marisol


    Full Text Available Abstract Background Double-stranded RNA (dsRNA is a potent initiator of gene silencing in a diverse group of organisms that includes plants, Caenorhabditis elegans, Drosophila and mammals. We have previously shown and patented that mechanical inoculation of in vitro-transcribed dsRNA derived from viral sequences specifically prevents virus infection in plants. The approach required the in vitro synthesis of large amounts of RNA involving high cost and considerable labour. Results We have developed an in vivo expression system to produce large amounts of virus-derived dsRNAs in bacteria, with a view to providing a practical control of virus diseases in plants. Partially purified bacterial dsRNAs promoted specific interference with the infection in plants by two viruses belonging to the tobamovirus and potyvirus groups. Furthermore, we have demonstrated that easy to obtain, crude extracts of bacterially expressed dsRNAs are equally effective protecting plants against virus infections when sprayed onto plant surfaces by a simple procedure. Virus infectivity was significantly abolished when plants were sprayed with French Press lysates several days before virus inoculation. Conclusion Our approach provides an alternative to genetic transformation of plant species with dsRNA-expressing constructs capable to interfere with plant viruses. The main advantage of this mode of dsRNA production is its simplicity and its extremely low cost compared with the requirements for regenerating transgenic plants. This approach provides a reliable and potential tool, not only for plant protection against virus diseases, but also for the study of gene silencing mechanisms in plant virus infections.

  1. An Effective Method for Extracting Total RNA from Young Embryo of Seedless Litchi%一种有效提取无核荔枝幼胚总RNA的方法

    Institute of Scientific and Technical Information of China (English)

    刘兴地; 郑学勤


    A total RNA extraction method for young embryo of seedless litchi was introduced.CRAB,Phenol(saturated with water),chloroform,Guanidine isothiecyanate were used as main extraction reagents.Polyphenolic compounds were removed effectively by added PVP into the extraction buffer solution.RNA was purified intensively by phenol,chloroform extraction,and ethanol deposition after deposited by LiCl.Both the results of formaldehyde denatured agarose gel electrophoresis and ultraviolet spectrophotometer analysis showed high integrity and purity of RNA.So the quality of extracted RNA could meet the demand of most molecular biology experiments that require higher quality RNA.

  2. A Suitable Method of RNA Extraction from Different Oil Palm Tissues%一种适合油棕不同组织 RNA 提取的方法

    Institute of Scientific and Technical Information of China (English)

    李静; 王永; 杨耀东; 雷新涛; 肖勇; 夏薇


    High quality of RNA is the important prerequisite and guarantee to basic research related to oil palm fruit development in molecular biology. RNA extracting effects of both Trizol reagents and MRIP (Methods for RNA Isolation from Palms) method developed by our laboratory previously were compared among four oil palm tissues, i.e. mature mesocarp, endosperm, embryo and germinated plumule. MRIP can get good quality RNA.The RNA content of plumule is as high as 2 139.0 ng/μL, while that of endosperm is only 1 083.6 ng/μL. MRIP is generally better than Trizol method in respect of the integrality, content and purity of extracted RNA, which Suitable for oil palm and palm plants RNA extraction.%获得高质量的 RNA 是开展油棕果实发育相关分子生物学基础研究的重要前提和保障。以油棕成熟果肉、胚乳、胚和胚芽4种组织为材料,分别采用 Invitrogen 公司的 Trizol 试剂和 MRIP 法提取总 RNA,并比较其提取效果。结果表明: MRIP 法提取的 RNA 质量较好,其中胚芽中的总 RNA 提取量高达2139.0 ng/μL,而胚乳中的含量相比较低,仅为1083.6 ng/μL; MRIP 法的总体提取效果优于 Trizol 试剂,包括 RNA 完整性、含量、纯度等,适用于油棕及棕榈科植物总 RNA 提取。

  3. Enhancement of single guide RNA transcription for efficient CRISPR/Cas-based genomic engineering. (United States)

    Ui-Tei, Kumiko; Maruyama, Shohei; Nakano, Yuko


    Genomic engineering using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein is a promising approach for targeting the genomic DNA of virtually any organism in a sequence-specific manner. Recent remarkable advances in CRISPR/Cas technology have made it a feasible system for use in therapeutic applications and biotechnology. In the CRISPR/Cas system, a guide RNA (gRNA), interacting with the Cas protein, recognizes a genomic region with sequence complementarity, and the double-stranded DNA at the target site is cleaved by the Cas protein. A widely used gRNA is an RNA polymerase III (pol III)-driven single gRNA (sgRNA), which is produced by artificial fusion of CRISPR RNA (crRNA) and trans-activation crRNA (tracrRNA). However, we identified a TTTT stretch, known as a termination signal of RNA pol III, in the scaffold region of the sgRNA. Here, we revealed that sgRNA carrying a TTTT stretch reduces the efficiency of sgRNA transcription due to premature transcriptional termination, and decreases the efficiency of genome editing. Unexpectedly, it was also shown that the premature terminated sgRNA may have an adverse effect of inducing RNA interference. Such disadvantageous effects were avoided by substituting one base in the TTTT stretch.

  4. Towards microRNA-based therapeutics for diabetic nephropathy. (United States)

    Alvarez, M L; DiStefano, J K


    There is no cure for diabetic nephropathy and the molecular mechanisms underlying disease aetiology remain poorly understood. While current paradigms for clinical management of diabetic nephropathy are useful in delaying disease onset and preventing its progression, they do not do so for a significant proportion of diabetic individuals, who eventually end up developing renal failure. Thus, novel therapeutic targets are needed for the treatment and prevention of the disease. MicroRNAs (miRNAs), a class of non-coding RNAs that negatively regulate gene expression, have recently been identified as attractive targets for therapeutic intervention. It is widely recognised that dysregulation of miRNA expression or action contributes to the development of a number of different human diseases, and evidence of a role for miRNAs in the aetiology of diabetic nephropathy is emerging. The discovery that modulation of miRNA expression in vivo is feasible, combined with recent results from successful clinical trials using this technology, opens the way for future novel therapeutic applications. For instance, inhibition of miRNAs that are commonly upregulated in diabetic nephropathy decreases albuminuria and mesangial matrix accumulation in animal models, suggesting that a therapeutic agent against these molecules may help to prevent the development of diabetic nephropathy. Certain challenges, including the development of safe and reliable delivery systems, remain to be overcome before miRNA-based therapeutics become a reality. However, the findings accumulated to date, in conjunction with newly emerging results, are expected to yield novel insights into the complex pathogenesis of diabetic nephropathy, and may eventually lead to the identification of improved therapeutic targets for treatment of this disease.

  5. Analytical Performances of Human Immunodeficiency Virus Type 1 RNA-Based Amplix® Real-Time PCR Platform for HIV-1 RNA Quantification

    Directory of Open Access Journals (Sweden)

    Christian Diamant Mossoro-Kpinde


    Full Text Available Objectives. We evaluated the performances of Amplix real-time PCR platform developed by Biosynex (Strasbourg, France, combining automated station extraction (Amplix station 16 Dx and real-time PCR (Amplix NG, for quantifying plasma HIV-1 RNA by lyophilized HIV-1 RNA-based Amplix reagents targeting gag and LTR, using samples from HIV-1-infected adults from Central African Republic. Results. Amplix real-time PCR assay showed low limit of detection (28 copies/mL, across wide dynamic range (1.4–10 log copies/mL, 100% sensitivity and 99% specificity, high reproducibility, and accuracy with mean bias < 5%. The assay showed excellent correlations and concordance of 95.3% with the reference HIV-1 RNA load assay (Roche, with mean absolute bias of +0.097 log copies/mL by Bland-Altman analysis. The assay was able to detect and quantify the most prevalent HIV-1 subtype strains and the majority of non-B subtypes, CRFs of HIV-1 group M, and HIV-1 groups N and O circulating in Central Africa. The Amplix assay showed 100% sensitivity and 99.6% specificity to diagnose virological failure in clinical samples from antiretroviral drug-experienced patients. Conclusions. The HIV-1 RNA-based Amplix real-time PCR platform constitutes sensitive and reliable system for clinical monitoring of HIV-1 RNA load in HIV-1-infected children and adults, particularly adapted to intermediate laboratory facilities in sub-Saharan Africa.

  6. Development of a flat membrane based device for electromembrane extraction

    DEFF Research Database (Denmark)

    Huang, Chuixiu; Eibak, Lars Erik Eng; Gjelstad, Astrid;


    In this work, a single-well electromembrane extraction (EME) device was developed based on a thin (100μm) and flat porous membrane of polypropylene supporting a liquid membrane. The new EME device was operated with a relatively large acceptor solution volume to promote a high recovery. Using...... for obtaining exhaustive extraction. 2-Nitrophenyl octyl ether was selected as the optimal organic solvent for the supported liquid membrane. From spiked acidified water samples (600μl), EME was carried out with 600μl of 20mM HCOOH as acceptor solution for 15min and with an extraction voltage of 250V. Under...... this EME device, exhaustive extraction of the basic drugs quetiapine, citalopram, amitriptyline, methadone and sertraline was investigated from both acidified water samples and human plasma. The volume of acceptor solution, extraction time, and extraction voltage were found to be important factors...

  7. An Improved TRIzol Method to Extract Total RNA from Skin Tissue of Rana dybowskii%改良TRIzol法高效提取东北林蛙皮肤总RNA

    Institute of Scientific and Technical Information of China (English)

    王丹丹; 肖向红; 吴立舒; 柴龙会; 张晶钰; 张建平


    Our objective was to develop a method for extracting high quality total RNA from amphibian skin tissue.We extracted total RNA from the skin of Dybowski's frog using a modified traditional TRIzol method.We assayed the purity and yield of the RNA by UV spectrophotometer.We examined the integrity of the extracted RNA by 1.1%agarose gel electrophoresis.The A260/A280 ratios of RNA extracted by our modified method ranged from 1.8 to 2.1,and the A260/A230 ratios were greater than 2.Agarose gel electrophoresis showed clear 28s rRNA and 18s rRNA bands,and the brightness of 28s rRNA bands was approximately twice the 18s rRNA.We conclude that the purity and integrity of total RNA extracted by our improved TRIzol method were higher than that extracted by the traditional TRIzol method:the total RNA had fewer impurities and the yields were larger.The modified method was easier to implement and can be used in relevant molecular biology tests.%目的:建立一种从两栖动物皮肤组织中提取高质量总RNA的方法。方法:改良传统TRIzol法,提取皮肤总RNA,紫外分光光度计测定RNA纯度和得率,1.1%琼脂糖凝胶电泳检测其完整性。结果:改良方法提取的总RNA,其A260/A280值在1.8~2.1之间,A260/A230大于2;琼脂糖凝胶电泳显示清晰的28s rRNA和18s rRNA条带,且28s rRNA条带亮度约为18s rRNA的2倍。结论:改良TRIzol法提取的总RNA纯度高、完整性好、杂质少、得率大,易于操作掌握,可以用于相应的分子生物学试验。

  8. Microarray analysis of RNA extracted from formalin-fixed, paraffin-embedded and matched fresh-frozen ovarian adenocarcinomas

    Directory of Open Access Journals (Sweden)

    Wu Thomas D


    Full Text Available Abstract Background Gene expression profiling of formalin-fixed, paraffin-embedded (FFPE samples represents a valuable approach for advancing oncology diagnostics and enhancing retrospective clinical studies; however, at present, this methodology still requires optimization and thus has not been extensively used. Here, we utilized thorough quality control methods to assess RNA extracted from FFPE samples and then compared it to RNA extracted from matched fresh-frozen (FF counterparts. We preformed genome-wide expression profiling of FF and FFPE ovarian serous adenocarcinoma sample pairs and compared their gene signatures to normal ovary samples. Methods RNA from FFPE samples was extracted using two different methods, Ambion and Agencourt, and its quality was determined by profiling starting total RNA on Bioanalyzer and by amplifying increasing size fragments of beta actin (ACTB and claudin 3 (CLDN3 by reverse-transcriptase polymerase chain reaction. Five matched FF and FFPE ovarian serous adenocarcinoma samples, as well as a set of normal ovary samples, were profiled using whole genome Agilent microarrays. Reproducibility of the FF and FFPE replicates was measured using Pearson correlation, whereas comparison between the FF and FFPE samples was done using a Z-score analysis. Results Data analysis showed high reproducibility of expression within each FF and FFPE method, whereas matched FF and FFPE pairs demonstrated lower similarity, emphasizing an inherent difference between the two sample types. Z-score analysis of matched FF and FFPE samples revealed good concordance of top 100 differentially expressed genes with the highest correlation of 0.84. Genes characteristic of ovarian serous adenocarcinoma, including a well known marker CLDN3, as well as potentially some novel markers, were identified by comparing gene expression profiles of ovarian adenocarcinoma to those of normal ovary. Conclusion Conclusively, we showed that systematic assessment

  9. Sponge-associated actinobacterial diversity: validation of the methods of actinobacterial DNA extraction and optimization of 16S rRNA gene amplification. (United States)

    Yang, Qi; Franco, Christopher M M; Zhang, Wei


    Experiments were designed to validate the two common DNA extraction protocols (CTAB-based method and DNeasy Blood & Tissue Kit) used to effectively recover actinobacterial DNA from sponge samples in order to study the sponge-associated actinobacterial diversity. This was done by artificially spiking sponge samples with actinobacteria (spores, mycelia and a combination of the two). Our results demonstrated that both DNA extraction methods were effective in obtaining DNA from the sponge samples as well as the sponge samples spiked with different amounts of actinobacteria. However, it was noted that in the presence of the sponge, the bacterial 16S rRNA gene could not be amplified unless the combined DNA template was diluted. To test the hypothesis that the extracted sponge DNA contained inhibitors, dilutions of the DNA extracts were tested for six sponge species representing five orders. The results suggested that the inhibitors were co-extracted with the sponge DNA, and a high dilution of this DNA was required for the successful PCR amplification for most of the samples. The optimized PCR conditions, including primer selection, PCR reaction system and program optimization, further improved the PCR performance. However, no single PCR condition was found to be suitable for the diverse sponge samples using various primer sets. These results highlight for the first time that the DNA extraction methods used are effective in obtaining actinobacterial DNA and that the presence of inhibitors in the sponge DNA requires high dilution coupled with fine tuning of the PCR conditions to achieve success in the study of sponge-associated actinobacterial diversity.

  10. New therapeutic opportunities for Hepatitis C based on small RNA

    Institute of Scientific and Technical Information of China (English)

    Qiu-wei Pan; Scot D Henry; Bob J Scholte; Hugo W Tilanus; Harry LA Janssen; Luc JW van der Laan


    Hepatitis C virus (HCV) infection is one of the major causes of chronic liver disease, including cirrhosis and liver cancer and is therefore, the most common indication for liver transplantation. Conventional antiviral drugs such as pegylated interferon-alpha, taken in combination with ribavirin, represent a milestone in the therapy of this disease. However, due to different viral and host factors, clinical success can be achieved only in approximately half of patients, making urgent the requirement of exploiting alternative approaches for HCV therapy. Fortunately, recent advances in the understanding of HCV viral replication and host cell interactions have opened new possibilities for therapeutic intervention. The most recent technologies, such as small interference RNA mediated gene-silencing, antisense oligonucleotides (ASO), or viral vector based gene delivery systems, have paved the way to develop novel therapeutic modalities for HCV. In this review, we outline the application of these technologies in the context of HCV therapy. Tn particular, we will focus on the newly defined role of cellular microRNA (miR-122) in viral replication and discuss its potential for HCV molecular therapy.

  11. An Effective History-based Background Extraction System

    Directory of Open Access Journals (Sweden)

    Fatimah Khalid


    Full Text Available Problem statement: In many visions-based surveillance systems, the first step is accomplished by detecting moving objects resulted from subtraction of the current captured frame from the extracted background. So, the results of these systems mainly depend on the accuracy of the background image. Approach: In this study, a proposed background extraction system is presented to model the background using a simple method, to initialize the model, to extract the moving objects and to construct the final background. Our model saves the history of each pixel separately. It uses the saved information to extract the background using a probability-based method. It updates the history of the pixel consequently and according to the value of that pixel in the current captured image. Results: Results of the experiments certify that not only the quality of the final extracted background is the best between four recently re-implemented methods, but also the time consumption of the extraction is acceptable. Conclusion: Since History-based methods use temporal information extracted from the several previous frames, they are less sensitive to noise and sudden changes for extracting the background image.

  12. RNA interference-based nanosystems for inflammatory bowel disease therapy

    Directory of Open Access Journals (Sweden)

    Guo J


    Full Text Available Jian Guo,1 Xiaojing Jiang,1 Shuangying Gui1,2 1Department of Pharmaceutics, College of Pharmacy, Anhui University of Chinese Medicine, 2Institute of Pharmaceutics, Anhui Academy of Chinese Medicine, Hefei, Anhui, People’s Republic of China Abstract: Inflammatory bowel disease (IBD, which includes ulcerative colitis and Crohn’s disease, is a chronic, recrudescent disease that invades the gastrointestinal tract, and it requires surgery or lifelong medicinal therapy. The conventional medicinal therapies for IBD, such as anti-inflammatories, glucocorticoids, and immunosuppressants, are limited because of their systemic adverse effects and toxicity during long-term treatment. RNA interference (RNAi precisely regulates susceptibility genes to decrease the expression of proinflammatory cytokines related to IBD, which effectively alleviates IBD progression and promotes intestinal mucosa recovery. RNAi molecules generally include short interfering RNA (siRNA and microRNA (miRNA. However, naked RNA tends to degrade in vivo as a consequence of endogenous ribonucleases and pH variations. Furthermore, RNAi treatment may cause unintended off-target effects and immunostimulation. Therefore, nanovectors of siRNA and miRNA were introduced to circumvent these obstacles. Herein, we introduce non-viral nanosystems of RNAi molecules and discuss these systems in detail. Additionally, the delivery barriers and challenges associated with RNAi molecules will be discussed from the perspectives of developing efficient delivery systems and potential clinical use. Keywords: RNA interference, siRNA, miRNA, nanoparticles, inflammatory bowel disease, target therapy

  13. Knowledge-based Approach for Event Extraction from Arabic Tweets

    Directory of Open Access Journals (Sweden)

    Mohammad AL-Smadi


    Full Text Available Tweets provide a continuous update on current events. However, Tweets are short, personalized and noisy, thus raises more challenges for event extraction and representation. Extracting events out of Arabic tweets is a new research domain where few examples – if any – of previous work can be found. This paper describes a knowledge-based approach for fostering event extraction out of Arabic tweets. The approach uses an unsupervised rule-based technique for event extraction and provides a named entity disambiguation of event related entities (i.e. person, organization, and location. Extracted events and their related entities are populated to the event knowledge base where tagged tweets’ entities are linked to their corresponding entities represented in the knowledge base. Proposed approach was evaluated on a dataset of 1K Arabic tweets covering different types of events (i.e. instant events and interval events. Results show that the approach has an accuracy of, 75.9% for event trigger extraction, 87.5% for event time extraction, and 97.7% for event type identification.

  14. Driver’s Fatigue Detection Based on Yawning Extraction

    Directory of Open Access Journals (Sweden)

    Nawal Alioua


    a real danger on road since it reduces driver capacity to react and analyze information. In this paper we propose an efficient and nonintrusive system for monitoring driver fatigue using yawning extraction. The proposed scheme uses face extraction based support vector machine (SVM and a new approach for mouth detection, based on circular Hough transform (CHT, applied on mouth extracted regions. Our system does not require any training data at any step or special cameras. Some experimental results showing system performance are reported. These experiments are applied over real video sequences acquired by low cost web camera and recorded in various lighting conditions.

  15. A Robust Digital Watermark Extracting Method Based on Neural Network

    Institute of Scientific and Technical Information of China (English)

    GUOLihua; YANGShutang; LIJianhua


    Since watermark removal software, such as StirMark, has succeeded in washing watermarks away for most of the known watermarking systems, it is necessary to improve the robustness of watermarking systems. A watermark extracting method based on the error Back propagation (BP) neural network is presented in this paper, which can efficiently improve the robustness of watermarking systems. Experiments show that even if the watermarking systems are attacked by the StirMark software, the extracting method based on neural network can still efficiently extract the whole watermark information.

  16. Extraction of Textual Causal Relationships based on Natural Language Processing

    Directory of Open Access Journals (Sweden)

    Sepideh Jamshidi-Nejad


    Full Text Available Natural language processing is a highly important subcategory in the wide area of artificial intelligence. Employing appropriate computational algorithms on sophisticated linguistic operations is the aim of natural language processing to extract and create computational theories from languages. In order to achieve this goal, the knowledge of linguists is needed in addition to computer science. In the field of linguistics, the syntactic and semantic relation of words and phrases and the extraction of causation is very significant which the latter is an information retrieval challenge. Recently, there is an increased attention towards the automatic extraction of causation from textual data sets. Although, previous research extracted the casual relations from uninterrupted data sets by using knowledge-based inference technologies and manual coding. Recently, finding comprehensive approaches for detection and extractions of causal arguments is a research area in the field of natural language processing.In this paper, a three-stepped approach is established through which, the position of words with syntax trees is obtained by extracting causation from causal and non-causal sentences of Web text. The arguments of events were extracted according to the dependency tree of phrases implemented by Python packages. Then potential causal relations were extracted by the extraction of specific nodes of the tree. In the final step, a statistical model is introduced for measuring the potential causal relations. Experimental results and evaluations with Recall, Precision and F-measure metrics show the accuracy and efficiency of the suggested model.

  17. Web entity extraction based on entity attribute classification (United States)

    Li, Chuan-Xi; Chen, Peng; Wang, Ru-Jing; Su, Ya-Ru


    The large amount of entity data are continuously published on web pages. Extracting these entities automatically for further application is very significant. Rule-based entity extraction method yields promising result, however, it is labor-intensive and hard to be scalable. The paper proposes a web entity extraction method based on entity attribute classification, which can avoid manual annotation of samples. First, web pages are segmented into different blocks by algorithm Vision-based Page Segmentation (VIPS), and a binary classifier LibSVM is trained to retrieve the candidate blocks which contain the entity contents. Second, the candidate blocks are partitioned into candidate items, and the classifiers using LibSVM are performed for the attributes annotation of the items and then the annotation results are aggregated into an entity. Results show that the proposed method performs well to extract agricultural supply and demand entities from web pages.

  18. End Sequence Analysis Toolkit (ESAT) expands the extractable information from single-cell RNA-seq data (United States)

    Derr, Alan; Yang, Chaoxing; Zilionis, Rapolas; Sergushichev, Alexey; Blodgett, David M.; Redick, Sambra; Bortell, Rita; Luban, Jeremy; Harlan, David M.; Kadener, Sebastian; Greiner, Dale L.; Klein, Allon; Artyomov, Maxim N.


    RNA-seq protocols that focus on transcript termini are well suited for applications in which template quantity is limiting. Here we show that, when applied to end-sequencing data, analytical methods designed for global RNA-seq produce computational artifacts. To remedy this, we created the End Sequence Analysis Toolkit (ESAT). As a test, we first compared end-sequencing and bulk RNA-seq using RNA from dendritic cells stimulated with lipopolysaccharide (LPS). As predicted by the telescripting model for transcriptional bursts, ESAT detected an LPS-stimulated shift to shorter 3′-isoforms that was not evident by conventional computational methods. Then, droplet-based microfluidics was used to generate 1000 cDNA libraries, each from an individual pancreatic islet cell. ESAT identified nine distinct cell types, three distinct β-cell types, and a complex interplay between hormone secretion and vascularization. ESAT, then, offers a much-needed and generally applicable computational pipeline for either bulk or single-cell RNA end-sequencing. PMID:27470110

  19. Alt Event Finder: a tool for extracting alternative splicing events from RNA-seq data


    Zhou Ao; Breese Marcus R; Hao Yangyang; Edenberg Howard J; Li Lang; Skaar Todd C; Liu Yunlong


    Abstract Background Alternative splicing increases proteome diversity by expressing multiple gene isoforms that often differ in function. Identifying alternative splicing events from RNA-seq experiments is important for understanding the diversity of transcripts and for investigating the regulation of splicing. Results We developed Alt Event Finder, a tool for identifying novel splicing events by using transcript annotation derived from genome-guided construction tools, such as Cufflinks and ...

  20. [Extraction of total RNA and cloning of sgDHAR gene from Siraitia grosvenorii]. (United States)

    Wei, Rong-Chang; Zhao, Huan; Ma, Xiao-Jun; Mi, Ke; Mo, Chang-Ming; Pan, Li-Mei; Bai, Long-Hua; Tang, Qi


    Total RNA was isolated from Siraitia grosvenorii fruit by the method of modified Trizol, according to S. grosvenorii fruit characteristics of rich phenols, polysaccharide, oil and proteins. The OD260/280, OD260/230, RNA integrity (RIN) and yield of the total RNA with this method were 2.01, 2.02, 9.50 and 260 mirog.g-1, respectively. The open reading frame (ORF) of dehydroascorbate reductase (DHAR), named as SgDHAR, was cloned by rapid amplification of cDNA ends (RACE) and RT-PCR method from S. grosvenorii. The GenBank accession number for this gene is KC907731. The SgDHAR gene contains a full-length cDNA of 1,252 bp including ORF of 819 bp and encodes a predicted protein of 272 amino acids. The molecular mass is 30.217 7 kD and the isoelectric point is 8.76. Homology comparison showed that it shared 87% nucleotide sequence homology with Cucumis sativus. Expression patterns using qRT-PCR analysis showed that SgDHAR was mainly expressed in fruit and stem, followed by flower, and was lowest in root, while the expression level was 6.83 times in triploid. T than that in diploid. Therefore, SgDHAR gene may be involved in abortion of triploid seedless S. grosvenorii.

  1. Efficient sparse kernel feature extraction based on partial least squares. (United States)

    Dhanjal, Charanpal; Gunn, Steve R; Shawe-Taylor, John


    The presence of irrelevant features in training data is a significant obstacle for many machine learning tasks. One approach to this problem is to extract appropriate features and, often, one selects a feature extraction method based on the inference algorithm. Here, we formalize a general framework for feature extraction, based on Partial Least Squares, in which one can select a user-defined criterion to compute projection directions. The framework draws together a number of existing results and provides additional insights into several popular feature extraction methods. Two new sparse kernel feature extraction methods are derived under the framework, called Sparse Maximal Alignment (SMA) and Sparse Maximal Covariance (SMC), respectively. Key advantages of these approaches include simple implementation and a training time which scales linearly in the number of examples. Furthermore, one can project a new test example using only k kernel evaluations, where k is the output dimensionality. Computational results on several real-world data sets show that SMA and SMC extract features which are as predictive as those found using other popular feature extraction methods. Additionally, on large text retrieval and face detection data sets, they produce features which match the performance of the original ones in conjunction with a Support Vector Machine.

  2. Predicting protein-RNA interaction amino acids using random forest based on submodularity subset selection. (United States)

    Pan, Xiaoyong; Zhu, Lin; Fan, Yong-Xian; Yan, Junchi


    Protein-RNA interaction plays a very crucial role in many biological processes, such as protein synthesis, transcription and post-transcription of gene expression and pathogenesis of disease. Especially RNAs always function through binding to proteins. Identification of binding interface region is especially useful for cellular pathways analysis and drug design. In this study, we proposed a novel approach for binding sites identification in proteins, which not only integrates local features and global features from protein sequence directly, but also constructed a balanced training dataset using sub-sampling based on submodularity subset selection. Firstly we extracted local features and global features from protein sequence, such as evolution information and molecule weight. Secondly, the number of non-interaction sites is much more than interaction sites, which leads to a sample imbalance problem, and hence biased machine learning model with preference to non-interaction sites. To better resolve this problem, instead of previous randomly sub-sampling over-represented non-interaction sites, a novel sampling approach based on submodularity subset selection was employed, which can select more representative data subset. Finally random forest were trained on optimally selected training subsets to predict interaction sites. Our result showed that our proposed method is very promising for predicting protein-RNA interaction residues, it achieved an accuracy of 0.863, which is better than other state-of-the-art methods. Furthermore, it also indicated the extracted global features have very strong discriminate ability for identifying interaction residues from random forest feature importance analysis.

  3. Behavior Based Social Dimensions Extraction for Multi-Label Classification. (United States)

    Li, Le; Xu, Junyi; Xiao, Weidong; Ge, Bin


    Classification based on social dimensions is commonly used to handle the multi-label classification task in heterogeneous networks. However, traditional methods, which mostly rely on the community detection algorithms to extract the latent social dimensions, produce unsatisfactory performance when community detection algorithms fail. In this paper, we propose a novel behavior based social dimensions extraction method to improve the classification performance in multi-label heterogeneous networks. In our method, nodes' behavior features, instead of community memberships, are used to extract social dimensions. By introducing Latent Dirichlet Allocation (LDA) to model the network generation process, nodes' connection behaviors with different communities can be extracted accurately, which are applied as latent social dimensions for classification. Experiments on various public datasets reveal that the proposed method can obtain satisfactory classification results in comparison to other state-of-the-art methods on smaller social dimensions.

  4. Study on Methods of Total RNA Extraction from Cotyledon of Chinese Chestnut%板栗子叶总RNA提取方法研究

    Institute of Scientific and Technical Information of China (English)

    王滨蔚; 车鹏燕; 何承忠; 王猛; 石卓功


    为了获得板栗子叶总RNA提取的理想方法,为后续的分子生物学研究打下基础,以板栗幼果子叶为材料,采用改良CTAB法、硼砂-CTAB法、酚-SDS法、皂土法和2种植物总RNA提取试剂盒共6种方法提取板栗子叶总RNA.结果表明,除硼砂-CTAB法外,其他5种方法均能获得板栗子叶总RNA.但是,与酚-SDS法、皂土法和2种植物总RNA提取试剂盒相比较,改良CTAB法提取的板栗子叶总RNA完整性好,纯度高,且无DNA污染,能够满足cDNA-AFLP等分子生物学研究的要求.%In order to obtain an ideal total RNA extraction method of cotyledon in Chinese chestnuts for providing foundation for molecular biology research, total RNA of cotyledon excised from young fruit of Chinese chestnut were extracted with 6 methods, including improved CTAB method, borax-CTAB method, phenol-SDS method, bentonite method and 2 RNA extraction kits. The results showed that total RNA was obtained with 5 methods but borax-CTAB method. However, the improved CTAB method was better than phenol-SDS method, bentonite method and two RNA extraction kits. The total RNA extracted with improved CTAB method had high integrity, high purity and no DNA pollution, and would be suitable for related molecular researches, such as cDNA-AFLP markers.

  5. Perceptual Object Extraction Based on Saliency and Clustering

    Directory of Open Access Journals (Sweden)

    Qiaorong Zhang


    Full Text Available Object-based visual attention has received an increasing interest in recent years. Perceptual object is the basic attention unit of object-based visual attention. The definition and extraction of perceptual objects is one of the key technologies in object-based visual attention computation model. A novel perceptual object definition and extraction method is proposed in this paper. Based on Gestalt theory and visual feature integration theory, perceptual object is defined using homogeneity region, salient region and edges. An improved saliency map generating algorithm is employed first. Based on the saliency map, salient edges are extracted. Then graph-based clustering algorithm is introduced to get homogeneity regions in the image. Finally an integration strategy is adopted to combine salient edges and homogeneity regions to extract perceptual objects. The proposed perceptual object extraction method has been tested on lots of natural images. Experiment results and analysis are presented in this paper also. Experiment results show that the proposed method is reasonable and valid.

  6. RNA interference-based nanosystems for inflammatory bowel disease therapy (United States)

    Guo, Jian; Jiang, Xiaojing; Gui, Shuangying


    Inflammatory bowel disease (IBD), which includes ulcerative colitis and Crohn’s disease, is a chronic, recrudescent disease that invades the gastrointestinal tract, and it requires surgery or lifelong medicinal therapy. The conventional medicinal therapies for IBD, such as anti-inflammatories, glucocorticoids, and immunosuppressants, are limited because of their systemic adverse effects and toxicity during long-term treatment. RNA interference (RNAi) precisely regulates susceptibility genes to decrease the expression of proinflammatory cytokines related to IBD, which effectively alleviates IBD progression and promotes intestinal mucosa recovery. RNAi molecules generally include short interfering RNA (siRNA) and microRNA (miRNA). However, naked RNA tends to degrade in vivo as a consequence of endogenous ribonucleases and pH variations. Furthermore, RNAi treatment may cause unintended off-target effects and immunostimulation. Therefore, nanovectors of siRNA and miRNA were introduced to circumvent these obstacles. Herein, we introduce non-viral nanosystems of RNAi molecules and discuss these systems in detail. Additionally, the delivery barriers and challenges associated with RNAi molecules will be discussed from the perspectives of developing efficient delivery systems and potential clinical use. PMID:27789943

  7. Design of guanidinium ionic liquid based microwave-assisted extraction for the efficient extraction of Praeruptorin A from Radix peucedani. (United States)

    Ding, Xueqin; Li, Li; Wang, Yuzhi; Chen, Jing; Huang, Yanhua; Xu, Kaijia


    A series of novel tetramethylguanidinium ionic liquids and hexaalkylguanidinium ionic liquids have been synthesized based on 1,1,3,3-tetramethylguanidine. The structures of the ionic liquids were confirmed by (1)H NMR spectroscopy and mass spectrometry. A green guanidinium ionic liquid based microwave-assisted extraction method has been developed with these guanidinium ionic liquids for the effective extraction of Praeruptorin A from Radix peucedani. After extraction, reversed-phase high-performance liquid chromatography with UV detection was employed for the analysis of Praeruptorin A. Several significant operating parameters were systematically optimized by single-factor and L9 (3(4)) orthogonal array experiments. The amount of Praeruptorin A extracted by [1,1,3,3-tetramethylguanidine]CH2CH(OH)COOH is the highest, reaching 11.05 ± 0.13 mg/g. Guanidinium ionic liquid based microwave-assisted extraction presents unique advantages in Praeruptorin A extraction compared with guanidinium ionic liquid based maceration extraction, guanidinium ionic liquid based heat reflux extraction and guanidinium ionic liquid based ultrasound-assisted extraction. The precision, stability, and repeatability of the process were investigated. The mechanisms of guanidinium ionic liquid based microwave-assisted extraction were researched by scanning electron microscopy and IR spectroscopy. All the results show that guanidinium ionic liquid based microwave-assisted extraction has a huge potential in the extraction of bioactive compounds from complex samples.

  8. Level Sets and Voronoi based Feature Extraction from any Imagery

    DEFF Research Database (Denmark)

    Sharma, O.; Anton, François; Mioc, Darka


    Polygon features are of interest in many GEOProcessing applications like shoreline mapping, boundary delineation, change detection, etc. This paper presents a unique new GPU-based methodology to automate feature extraction combining level sets, or mean shift based segmentation together with Voronoi...

  9. Phase extraction based on sinusoidal extreme strip phase shifting method (United States)

    Hui, Mei; Liu, Ming; Dong, Liquan; Liu, Xiaohua; Zhao, Yuejin


    Multiple synthetic aperture imaging can enlarge pupil diameter of optical systems, and increase system resolution. Multiple synthetic aperture imaging is a cutting-edge topic and research focus in recent years, which is prospectively widely applied in fields like astronomical observations and aerospace remote sensing. In order to achieve good imaging quality, synthetic aperture imaging system requires phase extraction of each sub-aperture and co-phasing of whole aperture. In the project, an in-depth study about basic principles and methods of segments phase extraction was done. The study includes: application of sinusoidal extreme strip light irradiation phase shift method to extract the central dividing line to get segment phase extraction information, and the use of interference measurement to get the aperture phase extraction calibration coefficients of spherical surface. Study about influence of sinusoidal extreme strip phase shift on phase extraction, and based on sinusoidal stripe phase shift from multiple linear light sources of the illumination reflected image, to carry out the phase shift error for inhibiting the effect in the phase extracted frame.

  10. Extraction of high quality of RNA and construction of a suppression subtractive hybridization (SSH) library from chestnut rose (Rosa roxburghii Tratt). (United States)

    Xu, Qiang; Wen, Xiaopeng; Tao, Nengguo; Hu, Zhiyong; Yue, Hailin; Deng, Xiuxin


    Chestnut rose (Rosa roxburghii Tratt) is a rare fruit crop of promising economical importance in fruit and ornamental exploitation in China. Isolation of high quality RNA from chestnut rose is difficult due to its high levels of polyphenols, polysaccharides and other compounds, but a modified CTAB extraction procedure without phenol gave satisfactory results. High concentrations of PVP (2%, w/v), CTAB (2%, w/v) and beta-mercaptoethanol (4%, v/v) were used in the extraction buffer to improve RNA quality. The average yield was about 200 microg RNA g(-1) fresh leaves. The isolated RNA was of sufficient quality for construction of suppression subtraction hybridization (SSH) library, which allowed the isolation of several pathogen-induced defense genes.

  11. Distinct summer and winter bacterial communities in the active layer of Svalbard permafrost revealed by DNA- and RNA-based analyses

    DEFF Research Database (Denmark)

    Schostag, Morten; Stibal, Marek; Jacobsen, Carsten S.;


    organic matter on the bacterial communities. The copy number of 16S rRNA genes and transcripts revealed no distinct seasonal changes indicating potential bacterial activity during winter despite soil temperatures well below -10ºC. Multivariate statistical analysis of the bacterial diversity data (DNA......N) by co-extracting DNA and RNA from 12 soil cores collected monthly over a year. PCR amplicons of 16S rRNA genes (DNA) and reverse transcribed transcripts (cDNA) were quantified and sequenced to test for the effect of low winter temperature and seasonal variation in concentration of easily degradable...... was significantly correlated with pH and dissolved organic carbon, while the potentially active (RNA-based) community structure was not significantly correlated with any of the measured soil parameters. A large fraction of the 16S rRNA transcripts was assigned to nitrogen-fixing bacteria (up to 24% in June...

  12. Algebraic characterization of RNA operations for DNA-based computation

    Institute of Scientific and Technical Information of China (English)

    LI Shuchao


    Any RNA strand can be presented by a word in the language X*, where X={A,C,G,U}. By encoding A as 010, C as 000, G as 111, and U as 101, the RNA operations can be considered as the performance of concatenation, union, reverse, complement, in terms of the algebraic characterization. The concatenation and union play the roles of multiplication and addition over some algebraic structures, respectively. The rest of the operations turn out to be the homomorphisms or anti-homomorphisms of these algebraic structures. Using this technique, we find the relationship among these RNA operations.

  13. Skeleton extraction based on the topology and Snakes model (United States)

    Cai, Yuanxue; Ming, Chengguo; Qin, Yueting

    A new skeleton line extraction method based on topology and flux is proposed by analyzing the distribution characteristics of the gradient vector field in the Snakes model. The distribution characteristics of the skeleton line are accurately obtained by calculating the eigenvalues of the critical points and the flux of the gradient vector field. Then the skeleton lines can be effectively extracted. The results also show that there is no need for the pretreatment or binarization of the target image. The skeleton lines of complex gray images such as optical interference patterns can be effectively extracted by using this method. Compared to traditional methods, this method has many advantages, such as high extraction accuracy and fast processing speed.

  14. Total RNA extraction of Ginkgo biloba leaves%银杏RNA的提取

    Institute of Scientific and Technical Information of China (English)

    程水源; 陈昆松; 杜何为; 许文平


    采用CTAB法提取银杏叶的RNA,结果表明:抽提的RNA经电泳检测,可见28 S rRNA和18 S rRNA两条主带;紫外吸收值测量,A260/A280接近2.0;反转录合成的cDNA经RAPD扩增,出现清晰的条带,这些说明提取的RNA纯度高,质量好.此方法简便易行,成本也较低,适合于富含多糖、多酚植物材料的RNA提取.

  15. Phylogenetic relationships of Salmonella based on rRNA sequences

    DEFF Research Database (Denmark)

    Christensen, H.; Nordentoft, Steen; Olsen, J.E.


    To establish the phylogenetic relationships between the subspecies of Salmonella enterica (official name Salmonella choleraesuis), Salmonella bongori and related members of Enterobacteriaceae, sequence comparison of rRNA was performed by maximum-likelihood analysis. The two Salmonella species wer...

  16. Extracting clinical information to support medical decision based on standards. (United States)

    Gomoi, Valentin; Vida, Mihaela; Stoicu-Tivadar, Lăcrămioara; Stoicu-Tivadar, Vasile


    The paper presents a method connecting medical databases to a medical decision system, and describes a service created to extract the necessary information that is transferred based on standards. The medical decision can be improved based on many inputs from different medical locations. The developed solution is described for a concrete case concerning the management for chronic pelvic pain, based on the information retrieved from diverse healthcare databases.

  17. Persistence of seed-based activity following segmentation of a microRNA guide strand. (United States)

    Chorn, Guillaume; Zhao, Lihong; Sachs, Alan B; Flanagan, W Michael; Lim, Lee P


    microRNAs are ∼ 22 nucleotide regulatory RNAs that are processed into duplexes from hairpin structures and incorporated into Argonaute proteins. Here, we show that a nick in the middle of the guide strand of an miRNA sequence allows for seed-based targeting characteristic of miRNA activity. Insertion of an inverted abasic, a dye, or a small gap between the two segments still permits target knockdown. While activity from the seed region of the segmented miRNA is apparent, activity from the 3' half of the guide strand is impaired, suggesting that an intact guide backbone is required for contribution from the 3' half. miRNA activity was also observed following nicking of a miRNA precursor. These results illustrate a structural flexibility in miRNA duplexes and may have applications in the design of miRNA mimetics.

  18. Messenger RNA vaccine based on recombinant MS2 virus-like particles against prostate cancer. (United States)

    Li, Jinming; Sun, Yanli; Jia, Tingting; Zhang, Rui; Zhang, Kuo; Wang, Lunan


    Prostate cancer (PCa) is the most diagnosed cancer in the western male population with high mortality. Recently, alternative approaches based on immunotherapy including mRNA vaccines for PCa have shown therapeutic promise. However, for mRNA vaccine, several disadvantages such as the instability of mRNA, the high cost of gold particles, the limited production scale for mRNA-transfected dendritic cells in vitro, limit their development. Herein, recombinant bacteriophage MS2 virus-like particles (VLPs), which based on the interaction of a 19-nucleotide RNA aptamer and the coat protein of bacteriophage MS2, successfully addressed these questions, in which target mRNA was packaged by MS2 capsid. MS2 VLP-based mRNA vaccines were easily prepared by recombinant protein technology, nontoxic and RNase-resistant. We show the packaged mRNA was translated into protein as early as 12 hr after phagocytosed by macrophages. Moreover, MS2 VLP-based mRNA vaccines induced strong humoral and cellular immune responses, especially antigen-specific cytotoxic T-lymphocyte (CTL) and balanced Th1/Th2 responses without upregulation of CD4(+) regulatory T cells, and protected C57BL/6 mice against PCa completely. As a therapeutic vaccine, MS2 VLP-based mRNA vaccines delayed tumor growth. Our results provide proof of concept on the efficacy and safety of MS2 VLP-based mRNA vaccine, which provides a new delivery approach for mRNA vaccine and implies important clinical value for the prevention and therapy of PCa.

  19. A Block-Based Multi-Scale Background Extraction Algorithm

    Directory of Open Access Journals (Sweden)

    Seyed H. Davarpanah


    Full Text Available Problem statement: To extract the moving objects, vision-based surveillance systems subtract the current image from a predefined background image. The efficiency of these systems mainly depends on accuracy of the extracted background image. It should be able to adapt to the changes continuously. In addition, especially in real-time applications the time complexity of this adaptation is a critical matter. Approach: In this study, to extract an adaptive background, a combination of blocking and multi-scale methods is presented. Because of being less sensitive to local movements, block-based techniques are proper to control the non-stationary objects’ movements, especially in outdoor applications. They can be useful to reduce the effect of these objects on the extracted background. We also used the blocking method to intelligently select the regions which the temporal filtering has to be applied on. In addition, an amended multi-scale algorithm is introduced. This algorithm is a hybrid algorithm, a combination of some nonparametric and parametric filters. It uses a nonparametric filter in the spatial domain to initiate two primary backgrounds. In continue two adapted two-dimensional filters will be used to extract the final background. Results: The qualitative and quantitative results of our experiments certify not only the quality of the final extracted background is acceptable, but also its time consumption is approximately half in compare to the similar methods. Conclusion: Using Multi scaling filtering and applying the filters just to some selected nonoverlapped blocks reduce the time consumption of the extracting background algorithm.

  20. DIANA-LncBase v2: indexing microRNA targets on non-coding transcripts. (United States)

    Paraskevopoulou, Maria D; Vlachos, Ioannis S; Karagkouni, Dimitra; Georgakilas, Georgios; Kanellos, Ilias; Vergoulis, Thanasis; Zagganas, Konstantinos; Tsanakas, Panayiotis; Floros, Evangelos; Dalamagas, Theodore; Hatzigeorgiou, Artemis G


    microRNAs (miRNAs) are short non-coding RNAs (ncRNAs) that act as post-transcriptional regulators of coding gene expression. Long non-coding RNAs (lncRNAs) have been recently reported to interact with miRNAs. The sponge-like function of lncRNAs introduces an extra layer of complexity in the miRNA interactome. DIANA-LncBase v1 provided a database of experimentally supported and in silico predicted miRNA Recognition Elements (MREs) on lncRNAs. The second version of LncBase ( presents an extensive collection of miRNA:lncRNA interactions. The significantly enhanced database includes more than 70 000 low and high-throughput, (in)direct miRNA:lncRNA experimentally supported interactions, derived from manually curated publications and the analysis of 153 AGO CLIP-Seq libraries. The new experimental module presents a 14-fold increase compared to the previous release. LncBase v2 hosts in silico predicted miRNA targets on lncRNAs, identified with the DIANA-microT algorithm. The relevant module provides millions of predicted miRNA binding sites, accompanied with detailed metadata and MRE conservation metrics. LncBase v2 caters information regarding cell type specific miRNA:lncRNA regulation and enables users to easily identify interactions in 66 different cell types, spanning 36 tissues for human and mouse. Database entries are also supported by accurate lncRNA expression information, derived from the analysis of more than 6 billion RNA-Seq reads.

  1. Extraction of Micronutrient Metals from Peat-based Media Using Various Chelate-ligand and Iron-source Extractants (United States)

    Objectives of the study were to determine effects of chelate-ligand (experiment 1) and iron-source (experiment 2) unbuffrered extractant solutions on substrate pH and Cu, Fe, Mn, and Zn extraction from peat-based media. Chelate-ligand extractants consisted of 5 mM solutions of ethylenediaminedisucc...

  2. SnapShot-Seq: a method for extracting genome-wide, in vivo mRNA dynamics from a single total RNA sample.

    Directory of Open Access Journals (Sweden)

    Jesse M Gray

    Full Text Available mRNA synthesis, processing, and destruction involve a complex series of molecular steps that are incompletely understood. Because the RNA intermediates in each of these steps have finite lifetimes, extensive mechanistic and dynamical information is encoded in total cellular RNA. Here we report the development of SnapShot-Seq, a set of computational methods that allow the determination of in vivo rates of pre-mRNA synthesis, splicing, intron degradation, and mRNA decay from a single RNA-Seq snapshot of total cellular RNA. SnapShot-Seq can detect in vivo changes in the rates of specific steps of splicing, and it provides genome-wide estimates of pre-mRNA synthesis rates comparable to those obtained via labeling of newly synthesized RNA. We used SnapShot-Seq to investigate the origins of the intrinsic bimodality of metazoan gene expression levels, and our results suggest that this bimodality is partly due to spillover of transcriptional activation from highly expressed genes to their poorly expressed neighbors. SnapShot-Seq dramatically expands the information obtainable from a standard RNA-Seq experiment.

  3. Tracking fungal community responses to maize plants by DNA- and RNA-based pyrosequencing.

    Directory of Open Access Journals (Sweden)

    Eiko E Kuramae

    Full Text Available We assessed soil fungal diversity and community structure at two sampling times (t1 = 47 days and t2 = 104 days of plant age in pots associated with four maize cultivars, including two genetically modified (GM cultivars by high-throughput pyrosequencing of the 18S rRNA gene using DNA and RNA templates. We detected no significant differences in soil fungal diversity and community structure associated with different plant cultivars. However, DNA-based analyses yielded lower fungal OTU richness as compared to RNA-based analyses. Clear differences in fungal community structure were also observed in relation to sampling time and the nucleic acid pool targeted (DNA versus RNA. The most abundant soil fungi, as recovered by DNA-based methods, did not necessary represent the most "active" fungi (as recovered via RNA. Interestingly, RNA-derived community compositions at t1 were highly similar to DNA-derived communities at t2, based on presence/absence measures of OTUs. We recovered large proportions of fungal sequences belonging to arbuscular mycorrhizal fungi and Basidiomycota, especially at the RNA level, suggesting that these important and potentially beneficial fungi are not affected by the plant cultivars nor by GM traits (Bt toxin production. Our results suggest that even though DNA- and RNA-derived soil fungal communities can be very different at a given time, RNA composition may have a predictive power of fungal community development through time.

  4. Improving information extraction using a probability-based approach

    DEFF Research Database (Denmark)

    Kim, S.; Ahmed, Saeema; Wallace, K.


    or retire. It is becoming essential to retrieve vital information from archived product documents, if it is available. There is, therefore, great interest in ways of extracting relevant and sharable information from documents. A keyword-based search is commonly used, but studies have shown...

  5. GARN: Sampling RNA 3D Structure Space with Game Theory and Knowledge-Based Scoring Strategies. (United States)

    Boudard, Mélanie; Bernauer, Julie; Barth, Dominique; Cohen, Johanne; Denise, Alain


    Cellular processes involve large numbers of RNA molecules. The functions of these RNA molecules and their binding to molecular machines are highly dependent on their 3D structures. One of the key challenges in RNA structure prediction and modeling is predicting the spatial arrangement of the various structural elements of RNA. As RNA folding is generally hierarchical, methods involving coarse-grained models hold great promise for this purpose. We present here a novel coarse-grained method for sampling, based on game theory and knowledge-based potentials. This strategy, GARN (Game Algorithm for RNa sampling), is often much faster than previously described techniques and generates large sets of solutions closely resembling the native structure. GARN is thus a suitable starting point for the molecular modeling of large RNAs, particularly those with experimental constraints. GARN is available from:

  6. Synthetic biology devices and circuits for RNA-based 'smart vaccines': a propositional review. (United States)

    Andries, Oliwia; Kitada, Tasuku; Bodner, Katie; Sanders, Niek N; Weiss, Ron


    Nucleic acid vaccines have been gaining attention as an alternative to the standard attenuated pathogen or protein based vaccine. However, an unrealized advantage of using such DNA or RNA based vaccination modalities is the ability to program within these nucleic acids regulatory devices that would provide an immunologist with the power to control the production of antigens and adjuvants in a desirable manner by administering small molecule drugs as chemical triggers. Advances in synthetic biology have resulted in the creation of highly predictable and modular genetic parts and devices that can be composed into synthetic gene circuits with complex behaviors. With the recent advent of modified RNA gene delivery methods and developments in the RNA replicon platform, we foresee a future in which mammalian synthetic biologists will create genetic circuits encoded exclusively on RNA. Here, we review the current repertoire of devices used in RNA synthetic biology and propose how programmable 'smart vaccines' will revolutionize the field of RNA vaccination.

  7. RIsearch2: suffix array-based large-scale prediction of RNA-RNA interactions and siRNA off-targets

    DEFF Research Database (Denmark)

    Alkan, Ferhat; Wenzel, Anne; Palasca, Oana


    and high level of complementarity between two RNA sequences is a powerful predictor of such interactions. Here, we present RIsearch2, a large-scale RNA-RNA interaction prediction tool that enables quick localization of potential near-complementary RNA-RNA interactions between given query and target...

  8. Effect of Green Tea Extract on Systemic Metabolic Homeostasis in Diet-Induced Obese Mice Determined via RNA-Seq Transcriptome Profiles (United States)

    Choi, Ji-Young; Kim, Ye Jin; Ryu, Ri; Cho, Su-Jung; Kwon, Eun-Young; Choi, Myung-Sook


    Green tea (GT) has various health effects, including anti-obesity properties. However, the multiple molecular mechanisms of the effects have not been fully determined. The aim of this study was to elucidate the anti-obesity effects of GT via the analysis of its metabolic and transcriptional responses based on RNA-seq profiles. C57BL/6J mice were fed a normal, high-fat (60% energy as fat), or high-fat + 0.25% (w/w) GT diet for 12 weeks. The GT extract ameliorated obesity, hepatic steatosis, dyslipidemia, and insulin resistance in diet-induced obesity (DIO) mice. GT supplementation resulted in body weight gain reduction than mice fed high-fat through enhanced energy expenditure, and reduced adiposity. The transcriptome profiles of epididymal white adipose tissue (eWAT) suggested that GT augments transcriptional responses to the degradation of branched chain amino acids (BCAAs), as well as AMP-activated protein kinase (AMPK) signaling, which suggests enhanced energy homeostasis. Our findings provide some significant insights into the effects of GT for the prevention of obesity and its comorbidities. We demonstrated that the GT extract contributed to the regulation of systemic metabolic homeostasis via transcriptional responses to not only lipid and glucose metabolism, but also amino acid metabolism via BCAA degradation in the adipose tissue of DIO mice. PMID:27754422

  9. Effect of Green Tea Extract on Systemic Metabolic Homeostasis in Diet-Induced Obese Mice Determined via RNA-Seq Transcriptome Profiles

    Directory of Open Access Journals (Sweden)

    Ji-Young Choi


    Full Text Available Green tea (GT has various health effects, including anti-obesity properties. However, the multiple molecular mechanisms of the effects have not been fully determined. The aim of this study was to elucidate the anti-obesity effects of GT via the analysis of its metabolic and transcriptional responses based on RNA-seq profiles. C57BL/6J mice were fed a normal, high-fat (60% energy as fat, or high-fat + 0.25% (w/w GT diet for 12 weeks. The GT extract ameliorated obesity, hepatic steatosis, dyslipidemia, and insulin resistance in diet-induced obesity (DIO mice. GT supplementation resulted in body weight gain reduction than mice fed high-fat through enhanced energy expenditure, and reduced adiposity. The transcriptome profiles of epididymal white adipose tissue (eWAT suggested that GT augments transcriptional responses to the degradation of branched chain amino acids (BCAAs, as well as AMP-activated protein kinase (AMPK signaling, which suggests enhanced energy homeostasis. Our findings provide some significant insights into the effects of GT for the prevention of obesity and its comorbidities. We demonstrated that the GT extract contributed to the regulation of systemic metabolic homeostasis via transcriptional responses to not only lipid and glucose metabolism, but also amino acid metabolism via BCAA degradation in the adipose tissue of DIO mice.

  10. Splicing of a C. elegans myosin pre-mRNA in a human nuclear extract

    Energy Technology Data Exchange (ETDEWEB)

    Ogg, S.C.; Anderson, P.; Wickens, M.P. (Univ. of Wisconsin, Madison (USA))


    Splicing of mammalian introns requires that the intron possess at least 80 nucleotides. This length requirement presumably reflects the constraints of accommodating multiple snRNPs simultaneously in the same intro. In the free-living nematode, C. elegans, introns typically are 45 to 55 nucleotides in length. In this report, the authors determine whether C. elegans introns can obviate the mammalian length requirement by virtue of their structure or sequence. They demonstrate that a 53 nucleotide intron from the unc-54 gene of C. elegans does not undergo splicing in a mammalian (HeLa) nuclear extract. However, insertion of 31 nucleotides of foreign, prokaryotic sequence into the same intron results in efficient splicing. The observed splicing proceeds by the same two-step mechanism observed with mammalian introns, and exploits the same 3{prime} and 5{prime} sites as are used in C. elegans. The branch point used lies in the inserted sequences. They conclude that C. elegans splicing components are either fewer in number or smaller than their mammalian counterparts.

  11. An assay for ribonuclease activity, based on ultraviolet absorption of RNA hydrolysate, using phosphotungstic acid. (United States)

    Isobe, K; Uchiyama, S


    In the method for the determination of ribonuclease activity that depends on the ultraviolet absorption of the RNA hydrolysate, the uranium reagent (25% perchloric acid solution containing 0.75% uranyl acetate) is commonly used for the efficient precipitation of the unhydrolyzed RNA. However, this reagent is always contaminated by the presence of radioactive isotopes. Radioactive uranium is one of the substances used for atomic nuclear fuel and therefore, at least in Japan, the use of uranium compounds requires permission from the government. We tried to find another efficient and non-radioactive precipitant of RNA to replace the uranium reagent, and have developed a phosphotungsten reagent (25% perchloric acid solution containing 0.75% phosphotungstic acid plus 0.6% bovine serum albumin solution) which functions as efficiently as the uranium reagent in the precipitation of RNA. A cell-free crude extract of Dictyostelium discoideum was used as the source of ribonuclease.

  12. Solvent extraction of gold using ionic liquid based process (United States)

    Makertihartha, I. G. B. N.; Zunita, Megawati; Rizki, Z.; Dharmawijaya, P. T.


    In decades, many research and mineral processing industries are using solvent extraction technology for metal ions separation. Solvent extraction technique has been used for the purification of precious metals such as Au and Pd, and base metals such as Cu, Zn and Cd. This process uses organic compounds as solvent. Organic solvents have some undesired properties i.e. toxic, volatile, excessive used, flammable, difficult to recycle, low reusability, low Au recovery, together with the problems related to the disposal of spent extractants and diluents, even the costs associated with these processes are relatively expensive. Therefore, a lot of research have boosted into the development of safe and environmentally friendly process for Au separation. Ionic liquids (ILs) are the potential alternative for gold extraction because they possess several desirable properties, such as a the ability to expanse temperature process up to 300°C, good solvent properties for a wide range of metal ions, high selectivity, low vapor pressures, stability up to 200°C, easy preparation, environmentally friendly (commonly called as "green solvent"), and relatively low cost. This review paper is focused in investigate of some ILs that have the potentials as solvent in extraction of Au from mineral/metal alloy at various conditions (pH, temperature, and pressure). Performances of ILs extraction of Au are studied in depth, i.e. structural relationship of ILs with capability to separate Au from metal ions aggregate. Optimal extraction conditon in order to gain high percent of Au in mineral processing is also investigated.

  13. Distributed Parallel Endmember Extraction of Hyperspectral Data Based on Spark

    Directory of Open Access Journals (Sweden)

    Zebin Wu


    Full Text Available Due to the increasing dimensionality and volume of remotely sensed hyperspectral data, the development of acceleration techniques for massive hyperspectral image analysis approaches is a very important challenge. Cloud computing offers many possibilities of distributed processing of hyperspectral datasets. This paper proposes a novel distributed parallel endmember extraction method based on iterative error analysis that utilizes cloud computing principles to efficiently process massive hyperspectral data. The proposed method takes advantage of technologies including MapReduce programming model, Hadoop Distributed File System (HDFS, and Apache Spark to realize distributed parallel implementation for hyperspectral endmember extraction, which significantly accelerates the computation of hyperspectral processing and provides high throughput access to large hyperspectral data. The experimental results, which are obtained by extracting endmembers of hyperspectral datasets on a cloud computing platform built on a cluster, demonstrate the effectiveness and computational efficiency of the proposed method.

  14. Approach to extracting hot topics based on network traffic content

    Institute of Scientific and Technical Information of China (English)

    Yadong ZHOU; Xiaohong GUAN; Qindong SUN; Wei LI; Jing TAO


    This article presents the formal definition and description of popular topics on the Internet,analyzes the relationship between popular words and topics,and finally introduces a method that uses statistics and correlation of the popular words in traffic content and network flow characteristics as input for extracting popular topics on the Internet.Based on this,this article adapts a clustering algorithm to extract popular topics and gives formalized results.The test results show that this method has an accuracy of 16.7% in extracting popular topics on the Internet.Compared with web mining and topic detection and tracking (TDT),it can provide a more suitable data source for effective recovery of Internet public opinions.

  15. Overlaid caption extraction in news video based on SVM (United States)

    Liu, Manman; Su, Yuting; Ji, Zhong


    Overlaid caption in news video often carries condensed semantic information which is key cues for content-based video indexing and retrieval. However, it is still a challenging work to extract caption from video because of its complex background and low resolution. In this paper, we propose an effective overlaid caption extraction approach for news video. We first scan the video key frames using a small window, and then classify the blocks into the text and non-text ones via support vector machine (SVM), with statistical features extracted from the gray level co-occurrence matrices, the LH and HL sub-bands wavelet coefficients and the orientated edge intensity ratios. Finally morphological filtering and projection profile analysis are employed to localize and refine the candidate caption regions. Experiments show its high performance on four 30-minute news video programs.

  16. Base pairing in RNA structures: A computational analysis of structural aspects and interaction energies

    Indian Academy of Sciences (India)

    Purshotam Sharma; Abhijit Mitra; Sitansh Sharma; Harjinder Singh


    The base pairing patterns in RNA structures are more versatile and completely different as compared to DNA. We present here results of ab-initio studies of structures and interaction energies of eight selected RNA base pairs reported in literature. Interaction energies, including BSSE correction, of hydrogen added crystal geometries of base pairs have been calculated at the HF/6-31G∗∗ level. The structures and interaction energies of the base pairs in the crystal geometry are compared with those obtained after optimization of the base pairs. We find that the base pairs become more planar on full optimization. No change in the hydrogen bonding pattern is seen. It is expected that the inclusion of appropriate considerations of many of these aspects of RNA base pairing would significantly improve the accuracy of RNA secondary structure prediction.

  17. siRNA delivery with lipid-based systems

    DEFF Research Database (Denmark)

    Foged, Camilla


    in vivo, toxicity and non-specific stimulation of the immune system. To optimally design and tailor the lipidic systems for siRNA delivery, better insight is needed into the mechanisms of cell delivery. More specifically, further clarification is need regarding the nature of cell surface interactions...

  18. miRNA-based drought regulation in wheat. (United States)

    Akdogan, Guray; Tufekci, Ebru Derelli; Uranbey, Serkan; Unver, Turgay


    MicroRNAs (miRNAs) are a class of small non-coding regulatory RNAs that regulate gene expression by guiding target mRNA cleavage or translational inhibition. Drought is a common environmental stress influencing crop growth and development. To date, it has been reported that a number of plant miRNA are involved in drought stress response. In this study, we comparatively investigated drought stress-responsive miRNAs in the root and leaf of bread wheat (Triticum aestivum cv. Sivas 111/33) by miRNA microarray screening. miRNA microarray analysis showed that 285 miRNAs (207 upregulated and 78 downregulated) and 244 miRNAs (115 upregulated and 129 downregulated) were differentially expressed in leaf and root tissues, respectively. Among the differentially expressed miRNAs, 23 miRNAs were only expressed in the leaf and 26 miRNAs were only expressed in the root of wheat growth under drought stress. Upon drought treatment, expression of miR159, miR160, miR166, miR169, miR172, miR395, miR396, miR408, miR472, miR477, miR482, miR1858, miR2118, and miR5049 were found to be significantly differentiated in bread wheat. The regulatory network analysis showed that miR395 has connections with a number of target transcripts, and miR159 and miR319 share a number of target genes. Drought-tolerant and drought-sensitive wheat cultivars showed altered expression pattern upon drought stress in terms of investigated miRNA and their target transcript expression level.

  19. Effects of Ganoderma lucidum (Higher Basidiomycetes) Extracts on the miRNA Profile and Telomerase Activity of the MCF-7 Breast Cancer Cell Line. (United States)

    Gonul, Oyku; Aydin, Hikmet Hakan; Kalmis, Erbil; Kayalar, Husniye; Ozkaya, Ali Burak; Atay, Sevcan; Ak, Handan


    Ganoderma lucidum is a medicinal higher Basidiomycetes mushroom that exerts anticancer effects through several different mechanisms. This study investigated the effects of G. lucidum on the telomerase activity and microRNA (miRNA) profiles of MCF-7 cells. According to the cytotoxicity results, the G. lucidum ether extract exhibits the highest cytotoxic potency; therefore it was chosen for the subsequent telomerase activity assay and miRNA profiling. The telomerase activity observed in the cells treated with a half-maximal inhibitory concentration of G. lucidum ether extract (100 µg/mL in dimethyl sulfoxide) was 32.2% lower than that of the control cells treated with 1% dimethyl sulfoxide. Among 1066 miRNAs, the most downregulated miRNA was hsa-miR-27a* (4.469-fold), and the most upregulated miRNA was hsa-miR-1285 (10.462-fold). A database search revealed the predicted miRNAs that target the catalytic subunit of the telomerase enzyme telomerase reverse transcriptase, and only miR-3687 (upregulated 2.153-fold) and miR-1207-5p (upregulated 2.895-fold) were changed by at least 2-fold. The miRNA profile changes demonstrated in this study provide a data set regarding their effects on the pathways that regulate telomerase activity in MCF-7 breast cancer cells treated with G. lucidum. These data should aid the development of novel cancer treatment strategies.

  20. Comprehensive protein-based artificial microRNA screens for effective gene silencing in plants. (United States)

    Li, Jian-Feng; Chung, Hoo Sun; Niu, Yajie; Bush, Jenifer; McCormack, Matthew; Sheen, Jen


    Artificial microRNA (amiRNA) approaches offer a powerful strategy for targeted gene manipulation in any plant species. However, the current unpredictability of amiRNA efficacy has limited broad application of this promising technology. To address this, we developed epitope-tagged protein-based amiRNA (ETPamir) screens, in which target mRNAs encoding epitope-tagged proteins were constitutively or inducibly coexpressed in protoplasts with amiRNA candidates targeting single or multiple genes. This design allowed parallel quantification of target proteins and mRNAs to define amiRNA efficacy and mechanism of action, circumventing unpredictable amiRNA expression/processing and antibody unavailability. Systematic evaluation of 63 amiRNAs in 79 ETPamir screens for 16 target genes revealed a simple, effective solution for selecting optimal amiRNAs from hundreds of computational predictions, reaching ∼100% gene silencing in plant cells and null phenotypes in transgenic plants. Optimal amiRNAs predominantly mediated highly specific translational repression at 5' coding regions with limited mRNA decay or cleavage. Our screens were easily applied to diverse plant species, including Arabidopsis thaliana, tobacco (Nicotiana benthamiana), tomato (Solanum lycopersicum), sunflower (Helianthus annuus), Catharanthus roseus, maize (Zea mays) and rice (Oryza sativa), and effectively validated predicted natural miRNA targets. These screens could improve plant research and crop engineering by making amiRNA a more predictable and manageable genetic and functional genomic technology.

  1. RNA-Based Methods Increase the Detection of Fecal Bacteria and Fecal Identifiers in Environmental Waters (United States)

    We evaluated the use of qPCR RNA-based methods in the detection of fecal bacteria in environmental waters. We showed that RNA methods can increase the detection of fecal bacteria in multiple water matrices. The data suggest that this is a viable alternative for the detection of a...

  2. Enhanced Detection of Bacteria in Environmental Waters: an RNA-based Approach (United States)

    Molecular assays (i.e., PCR and qPCR) used in microbial water quality studies often target ribosomal RNA genes (rDNA). However, using DNA as the PCR template does not discriminate between active and dead cells. The use of RNA-based detection methods has recently been proposed as ...

  3. RNA aptamer-based electrochemical biosensor for selective and label-free analysis of dopamine

    DEFF Research Database (Denmark)

    Farjami, Elahe; Campos, Rui; Nielsen, Jesper Sejrup


    , including dopamine precursors and metabolites and other neurotransmitters (NT). Here we report an electrochemical RNA aptamer-based biosensor for analysis of dopamine in the presence of other NT. The biosensor exploits a specific binding of dopamine by the RNA aptamer, immobilized at a cysteamine...

  4. An Improved CTAB Method for Extraction of Total RNA from Mature Leaves in Oil Sunflower%改良CTAB法提取油用向日葵成熟叶片的总RNA

    Institute of Scientific and Technical Information of China (English)


      为从植物中提取纯度高、完整性好的RNA ,采用改良过的CTAB法提取油用向日葵改 HA89开花后12 d成熟叶片的总RNA ,并对其进行了紫外分光光度计和琼脂糖凝胶电泳分析。结果表明:得到的RNA完整性好,纯度和浓度均较高,可用于后续的RT-PCR、Northern杂交和cDNA文库的构建等分子生物学实验研究。%In order to extract RNA of high purity and good integrity from plants ,after 12 days’ flowering the total RNA from mature leaves in oil sunflower Modified HA 89 was extracted through improved CTAB meth-od .It was analyzed by UV spectrophotometer and agarose gel electrophoresis .The results showed that the structure of total RNA obtained was complete with good purity and high concentration .It could be used for subsequent experiments of molecular biology such as RT-PCR ,Northern hybridization ,cDNA library construction .

  5. Multiplexing seven miRNA-Based shRNAs to suppress HIV replication. (United States)

    Choi, Jang-Gi; Bharaj, Preeti; Abraham, Sojan; Ma, Hongming; Yi, Guohua; Ye, Chunting; Dang, Ying; Manjunath, N; Wu, Haoquan; Shankar, Premlata


    Multiplexed miRNA-based shRNAs (shRNA-miRs) could have wide potential to simultaneously suppress multiple genes. Here, we describe a simple strategy to express a large number of shRNA-miRs using minimal flanking sequences from multiple endogenous miRNAs. We found that a sequence of 30 nucleotides flanking the miRNA duplex was sufficient for efficient processing of shRNA-miRs. We inserted multiple shRNAs in tandem, each containing minimal flanking sequence from a different miRNA. Deep sequencing of transfected cells showed accurate processing of individual shRNA-miRs and that their expression did not decrease with the distance from the promoter. Moreover, each shRNA was as functionally competent as its singly expressed counterpart. We used this system to express one shRNA-miR targeting CCR5 and six shRNA-miRs targeting the HIV-1 genome. The lentiviral construct was pseudotyped with HIV-1 envelope to allow transduction of both resting and activated primary CD4 T cells. Unlike one shRNA-miR, the seven shRNA-miR transduced T cells nearly abrogated HIV-1 infection in vitro. Additionally, when PBMCs from HIV-1 seropositive individuals were transduced and transplanted into NOD/SCID/IL-2R γc(-/-) mice (Hu-PBL model) efficient suppression of endogenous HIV-1 replication with restoration of CD4 T cell counts was observed. Thus, our multiplexed shRNA appears to provide a promising gene therapeutic approach for HIV-1 infection.

  6. 两种同时提取小麦根腐离蠕孢菌DNA和RNA的方法比较%Comparing Two Methods for Simultaneous Extraction of DNA and RNA for Bipolaris sorokinianum

    Institute of Scientific and Technical Information of China (English)

    王珍; 崔素萍; 崔丽琴; 左豫虎


    Obtaining sufficient quantity and high quality of RNA and DNA is the base for carrying out researches in molecular biology. DNA from Bipolaris sorokiniana was attracted by two cell wall lysis methods using lysozyme and liquid nitrogen grinding methods, respectively. Integrality and purity of nucleic acids were detected with agarose gel electrophoresis, ultraviolet absorption and PCR. Ideal results were obtained, when extracting DNA, high quality RNA was obtained at the same time, by using both methods. The results of PCR indicated that the integrality of DNA was good, The quality of DNA was similar for the two methods used, while the quality of RNA was better when extracted with lysozyme than it with liquid nitrogen grinding. The DNA extraction system of Bipolaris sorokiniana by using lysozyme method was suitable for RNA extraction, it provided a simple, safe and feasible method for extracting both DNA and RNA at the same time from Bipolaris sorokiniana.%获得足够数量和高质量的RNA和DNA是进行分子生物学研究工作的基础.以小麦根腐离蠕孢菌为原料,分别采用溶菌酶法和液氮研磨法破碎真菌细胞壁提取DNA,用琼脂糖凝胶电泳、紫外吸收及聚合酶链式反应(PCR)技术进行核酸完整性和纯度检测.结果表明:2种方法在提取DNA时都同时得到了高质量的RNA;PCR检测结果表明DNA完整性比较高,2种方法得到的DNA质量相当;溶菌酶法中得到的RNA质量优于液氮法.利用溶菌酶破壁法提取小麦根腐离蠕孢菌DNA的提取体系可用于RNA的提取,为小麦根腐离蠕孢菌核酸的DNA和RNA的同时提取提供了一种简便、安全、可行的方法.

  7. Organocatalytic Removal of Formaldehyde Adducts from RNA and DNA Bases



    Formaldehyde is universally employed to fix tissue specimens, where it forms hemiaminal and aminal adducts with biomolecules, hindering the ability to retrieve molecular information. Common methods for removing these adducts involve extended heating, which can cause extensive degradation of nucleic acids, particularly RNA. Here we show that water-soluble bifunctional catalysts (anthranilates and phosphanilates) speed the reversal of formaldehyde adducts of mononucleotides over standard buffer...

  8. Curvelet Transform-Based Denoising Method for Doppler Frequency Extraction

    Institute of Scientific and Technical Information of China (English)

    HOU Shu-juan; WU Si-liang


    A novel image denoising method based on curvelet transform is proposed in order to improve the performance of Doppler frequency extraction in low signal-noise-ratio (SNR) environment. The echo can be represented as a gray image with spectral intensity as its gray values by time-frequency transform. And the curvelet coefficients of the image are computed. Then an adaptive soft-threshold scheme based on dual-median operation is implemented in curvelet domain. After that, the image is reconstructed by inverse curvelet transform and the Doppler curve is extracted by a curve detection scheme. Experimental results show the proposed method can improve the detection of Doppler frequency in low SNR environment.

  9. Feature Extraction based Face Recognition, Gender and Age Classification

    Directory of Open Access Journals (Sweden)

    Venugopal K R


    Full Text Available The face recognition system with large sets of training sets for personal identification normally attains good accuracy. In this paper, we proposed Feature Extraction based Face Recognition, Gender and Age Classification (FEBFRGAC algorithm with only small training sets and it yields good results even with one image per person. This process involves three stages: Pre-processing, Feature Extraction and Classification. The geometric features of facial images like eyes, nose, mouth etc. are located by using Canny edge operator and face recognition is performed. Based on the texture and shape information gender and age classification is done using Posteriori Class Probability and Artificial Neural Network respectively. It is observed that the face recognition is 100%, the gender and age classification is around 98% and 94% respectively.

  10. Prediction of viral microRNA precursors based on human microRNA precursor sequence and structural features. (United States)

    Kumar, Shiva; Ansari, Faraz A; Scaria, Vinod


    MicroRNAs (small approximately 22 nucleotide long non-coding endogenous RNAs) have recently attracted immense attention as critical regulators of gene expression in multi-cellular eukaryotes, especially in humans. Recent studies have proved that viruses also express microRNAs, which are thought to contribute to the intricate mechanisms of host-pathogen interactions. Computational predictions have greatly accelerated the discovery of microRNAs. However, most of these widely used tools are dependent on structural features and sequence conservation which limits their use in discovering novel virus expressed microRNAs and non-conserved eukaryotic microRNAs. In this work an efficient prediction method is developed based on the hypothesis that sequence and structure features which discriminate between host microRNA precursor hairpins and pseudo microRNAs are shared by viral microRNA as they depend on host machinery for the processing of microRNA precursors. The proposed method has been found to be more efficient than recently reported ab-initio methods for predicting viral microRNAs and microRNAs expressed by mammals.

  11. Green tea extract increases cyclophosphamide-induced teratogenesis by modulating the expression of cytochrome P-450 mRNA. (United States)

    Park, Dongsun; Jeon, Jeong Hee; Shin, Sunhee; Joo, Seong Soo; Kang, Dae-Hyuck; Moon, Seol-Hee; Jang, Min-Jung; Cho, Yeoung Mi; Kim, Jae Wook; Ji, Hyeong-Jin; Ahn, Byeongwoo; Oh, Ki-Wan; Kim, Yun-Bae


    The effects of green tea extract (GTE) on the fetal development and external, visceral and skeletal abnormalities induced by cyclophosphamide were investigated in rats. Pregnant rats were daily administered GTE (100mg/kg) by gavage for 7 d, from the 6th to 12th day of gestation, and intraperitoneally administered with cyclophosphamide (11mg/kg) 1h after the final treatment. On the 20th day of gestation, maternal and fetal abnormalities were determined by Cesarian section. Cyclophosphamide was found to reduce fetal and placental weights without increasing resorption or death. In addition, it induced malformations in live fetuses; 94.6%, 41.5% and 100% of the external (skull and limb defects), visceral (cleft palate and ureteric dilatation) and skeletal (acrania, vertebral/costal malformations and delayed ossification) abnormalities. When pre-treated with GTE, cyclophosphamide-induced body weight loss and abnormalities of fetuses were remarkably aggravated. Moreover, repeated treatment with GTE greatly increased mRNA expression and activity of hepatic cytochrome P-450 (CYP) 2B, which metabolizes cyclophosphamide into teratogenic acrolein and cytotoxic phosphoramide mustard, while reducing CYP3A expression (a detoxifying enzyme). The results suggest that repeated intake of GTE may aggravate cyclophosphamide-induced body weight loss and malformations of fetuses by modulating CYP2B and CYP3A.

  12. Evaluating a Pivot-Based Approach for Bilingual Lexicon Extraction

    Directory of Open Access Journals (Sweden)

    Jae-Hoon Kim


    Full Text Available A pivot-based approach for bilingual lexicon extraction is based on the similarity of context vectors represented by words in a pivot language like English. In this paper, in order to show validity and usability of the pivot-based approach, we evaluate the approach in company with two different methods for estimating context vectors: one estimates them from two parallel corpora based on word association between source words (resp., target words and pivot words and the other estimates them from two parallel corpora based on word alignment tools for statistical machine translation. Empirical results on two language pairs (e.g., Korean-Spanish and Korean-French have shown that the pivot-based approach is very promising for resource-poor languages and this approach observes its validity and usability. Furthermore, for words with low frequency, our method is also well performed.

  13. Large-Scale Investigation of Human TF-miRNA Relations Based on Coexpression Profiles

    Directory of Open Access Journals (Sweden)

    Chia-Hung Chien


    Full Text Available Noncoding, endogenous microRNAs (miRNAs are fairly well known for regulating gene expression rather than protein coding. Dysregulation of miRNA gene, either upregulated or downregulated, may lead to severe diseases or oncogenesis, especially when the miRNA disorder involves significant bioreactions or pathways. Thus, how miRNA genes are transcriptionally regulated has been highlighted as well as target recognition in recent years. In this study, a large-scale investigation of novel cis- and trans-elements was undertaken to further determine TF-miRNA regulatory relations, which are necessary to unravel the transcriptional regulation of miRNA genes. Based on miRNA and annotated gene expression profiles, the term “coTFBS” was introduced to detect common transcription factors and the corresponding binding sites within the promoter regions of each miRNA and its coexpressed annotated genes. The computational pipeline was successfully established to filter redundancy due to short sequence motifs for TFBS pattern search. Eventually, we identified more convinced TF-miRNA regulatory relations for 225 human miRNAs. This valuable information is helpful in understanding miRNA functions and provides knowledge to evaluate the therapeutic potential in clinical research. Once most expression profiles of miRNAs in the latest database are completed, TF candidates of more miRNAs can be explored by this filtering approach in the future.

  14. Isolation of Microarray-Grade Total RNA, MicroRNA, and DNA from a Single PAXgene Blood RNA Tube

    DEFF Research Database (Denmark)

    Kruhøffer, Mogens; Andersen, Lars Dyrskjøt; Voss, Thorsten;


    We have developed a procedure for isolation of microRNA and genomic DNA in addition to total RNA from whole blood stabilized in PAXgene Blood RNA tubes. The procedure is based on automatic extraction on a BioRobot MDx and includes isolation of DNA from a fraction of the stabilized blood...... and recovery of small RNA species that are otherwise lost. The procedure presented here is suitable for large-scale experiments and is amenable to further automation. Procured total RNA and DNA was tested using Affymetrix Expression and single-nucleotide polymorphism GeneChips, respectively, and isolated micro......RNA was tested using spotted locked nucleic acid-based microarrays. We conclude that the yield and quality of total RNA, microRNA, and DNA from a single PAXgene blood RNA tube is sufficient for downstream microarray analysis....

  15. Arduino-based automation of a DNA extraction system. (United States)

    Kim, Kyung-Won; Lee, Mi-So; Ryu, Mun-Ho; Kim, Jong-Won


    There have been many studies to detect infectious diseases with the molecular genetic method. This study presents an automation process for a DNA extraction system based on microfluidics and magnetic bead, which is part of a portable molecular genetic test system. This DNA extraction system consists of a cartridge with chambers, syringes, four linear stepper actuators, and a rotary stepper actuator. The actuators provide a sequence of steps in the DNA extraction process, such as transporting, mixing, and washing for the gene specimen, magnetic bead, and reagent solutions. The proposed automation system consists of a PC-based host application and an Arduino-based controller. The host application compiles a G code sequence file and interfaces with the controller to execute the compiled sequence. The controller executes stepper motor axis motion, time delay, and input-output manipulation. It drives the stepper motor with an open library, which provides a smooth linear acceleration profile. The controller also provides a homing sequence to establish the motor's reference position, and hard limit checking to prevent any over-travelling. The proposed system was implemented and its functionality was investigated, especially regarding positioning accuracy and velocity profile.

  16. Facial Feature Extraction Method Based on Coefficients of Variances

    Institute of Scientific and Technical Information of China (English)

    Feng-Xi Song; David Zhang; Cai-Kou Chen; Jing-Yu Yang


    Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA) are two popular feature ex- traction techniques in statistical pattern recognition field. Due to small sample size problem LDA cannot be directly applied to appearance-based face recognition tasks. As a consequence, a lot of LDA-based facial feature extraction techniques are proposed to deal with the problem one after the other. Nullspace Method is one of the most effective methods among them. The Nullspace Method tries to find a set of discriminant vectors which maximize the between-class scatter in the null space of the within-class scatter matrix. The calculation of its discriminant vectors will involve performing singular value decomposition on a high-dimensional matrix. It is generally memory- and time-consuming. Borrowing the key idea in Nullspace method and the concept of coefficient of variance in statistical analysis we present a novel facial feature extraction method, i.e., Discriminant based on Coefficient of Variance (DCV) in this paper. Experimental results performed on the FERET and AR face image databases demonstrate that DCV is a promising technique in comparison with Eigenfaces, Nullspace Method, and other state-of-the-art facial feature extraction methods.

  17. Gut Microbiota Analysis Results Are Highly Dependent on the 16S rRNA Gene Target Region, Whereas the Impact of DNA Extraction Is Minor (United States)

    Rintala, Anniina; Pietilä, Sami; Munukka, Eveliina; Eerola, Erkki; Pursiheimo, Juha-Pekka; Laiho, Asta; Pekkala, Satu; Huovinen, Pentti


    Next-generation sequencing (NGS) is currently the method of choice for analyzing gut microbiota composition. As gut microbiota composition is a potential future target for clinical diagnostics, it is of utmost importance to enhance and optimize the NGS analysis procedures. Here, we have analyzed the impact of DNA extraction and selected 16S rDNA primers on the gut microbiota NGS results. Bacterial DNA from frozen stool specimens was extracted with 5 commercially available DNA extraction kits. Special attention was paid to the semiautomated DNA extraction methods that could expedite the analysis procedure, thus being especially suitable for clinical settings. The microbial composition was analyzed with 2 distinct protocols: 1 targeting the V3–V4 and the other targeting the V4–V5 area of the bacterial 16S rRNA gene. The overall effect of DNA extraction on the gut microbiota 16S rDNA profile was relatively small, whereas the 16S rRNA gene target region had an immense impact on the results. Furthermore, semiautomated DNA extraction methods clearly appeared suitable for NGS procedures, proposing that application of these methods could importantly reduce hands-on time and human errors without compromising the validity of results. PMID:28260999

  18. Establishment of a rapid, inexpensive protocol for extraction of high quality RNA from small amounts of strawberry plant tissues and other recalcitrant fruit crops. (United States)

    Christou, Anastasis; Georgiadou, Egli C; Filippou, Panagiota; Manganaris, George A; Fotopoulos, Vasileios


    Strawberry plant tissues and particularly fruit material are rich in polysaccharides and polyphenolic compounds, thus rendering the isolation of nucleic acids a difficult task. This work describes the successful modification of a total RNA extraction protocol, which enables the isolation of high quantity and quality of total RNA from small amounts of strawberry leaf, root and fruit tissues. Reverse-transcription polymerase chain reaction (RT-PCR) amplification of GAPDH housekeeping gene from isolated RNA further supports the proposed protocol efficiency and its use for downstream molecular applications. This novel procedure was also successfully followed using other fruit tissues, such as olive and kiwifruit. In addition, optional treatment with RNase A following initial nucleic acid extraction can provide sufficient quality and quality of genomic DNA for subsequent PCR analyses, as evidenced from PCR amplification of housekeeping genes using extracted genomic DNA as template. Overall, this optimized protocol allows easy, rapid and economic isolation of high quality RNA from small amounts of an important fruit crop, such as strawberry, with extended applicability to other recalcitrant fruit crops.

  19. Comparison of Two RNA Extraction Methods of Coconut Tree%2种提取椰子树RNA方法的比较研究

    Institute of Scientific and Technical Information of China (English)

    杨光华; 刘进平


    椰子树组织中富含多糖和酚类化合物,为了获取高质量的RNA,笔者以椰子树叶片和根部为材料,比较分析CTAB法和SDS法提取RNA的效果。结果表明:CTAB法提取椰子树组织的总RNA效果较好,能有效克服酚类物质和多糖对RNA提取的影响。经OD值检测和电泳检测,总RNA的28 S和18 S条带清晰可见,纯度较高,完整性好,无明显降解,以此RNA为模板进行RT-PCR反应,能获得特异条带,表明该RNA完全可以用于后续的分子生物学操作。%Tissue of coconut tree is rich in polysaccharides and phenolic compounds.In order to obtain high-quality RNA,comparison of SDS method and CTAB method for total RNA extraction from leaves and roots of coconut tree was conducted.The results showed that CTAB method was more suitable and effective than SDS method in the total RNA extraction of coconut trees and effectively overcame the problems caused by polyphenols and polysaccharides in the RNA extraction.As detected with OD value and gel electrophoresis,bands of 28S and 18S rRNA are clearly visible,with high purity,integrity and no degradation.With the RNAs as templates for RT-PCR reaction,specific bands were produced,indicating that the RNAs could be used for subsequent molecular biological operations.

  20. Does base-pairing strength play a role in microRNA repression?



    In this paper, the authors explore the possibility that the base-pairing strength of miRNA–mRNA target plays a role in microRNA-mediated gene regulation. The authors found a significant correlation between the physiological temperature of the organism and the average G/C content of its miRNAs. This study suggests that an organism adapts its miRNA–target free energy according to its physiological temperature, thus highlighting the importance of base-pairing strength in miRNA activity.

  1. End Sequence Analysis Toolkit (ESAT) expands the extractable information from single-cell RNA-seq data


    Derr, Alan; Yang, Chaoxing; Žilionis, Rapolas; Sergushichev, Alexey; Blodgett, David M.; Redick, Sambra; Bortell, Rita; Luban, Jeremy; Harlan, David M.; Kadener, Sebastian; Greiner, Dale L.; Klein, Allon; Artyomov, Maxim N.; Garber, Manuel


    RNA-seq protocols that focus on transcript termini are well suited for applications in which template quantity is limiting. Here we show that, when applied to end-sequencing data, analytical methods designed for global RNA-seq produce computational artifacts. To remedy this, we created the End Sequence Analysis Toolkit (ESAT). As a test, we first compared end-sequencing and bulk RNA-seq using RNA from dendritic cells stimulated with lipopolysaccharide (LPS). As predicted by the telescripting ...

  2. The Influence of DNA Extraction Procedure and Primer Set on the Bacterial Community Analysis by Pyrosequencing of Barcoded 16S rRNA Gene Amplicons. (United States)

    Starke, Ingo C; Vahjen, Wilfried; Pieper, Robert; Zentek, Jürgen


    In this study, the effect of different DNA extraction procedures and primer sets on pyrosequencing results regarding the composition of bacterial communities in the ileum of piglets was investigated. Ileal chyme from piglets fed a diet containing different amounts of zinc oxide was used to evaluate a pyrosequencing study with barcoded 16S rRNA PCR products. Two DNA extraction methods (bead beating versus silica gel columns) and two primer sets targeting variable regions of bacterial 16S rRNA genes (8f-534r versus 968f-1401r) were considered. The SEED viewer software of the MG-RAST server was used for automated sequence analysis. A total of 5.2 × 10(5) sequences were used for analysis after processing for read length (150 bp), minimum sequence occurrence (5), and exclusion of eukaryotic and unclassified/uncultured sequences. DNA extraction procedures and primer sets differed significantly in total sequence yield. The distribution of bacterial order and main bacterial genera was influenced significantly by both parameters. However, this study has shown that the results of pyrosequencing studies using barcoded PCR amplicons of bacterial 16S rRNA genes depend on DNA extraction and primer choice, as well as on the manner of downstream sequence analysis.

  3. The Influence of DNA Extraction Procedure and Primer Set on the Bacterial Community Analysis by Pyrosequencing of Barcoded 16S rRNA Gene Amplicons

    Directory of Open Access Journals (Sweden)

    Ingo C. Starke


    Full Text Available In this study, the effect of different DNA extraction procedures and primer sets on pyrosequencing results regarding the composition of bacterial communities in the ileum of piglets was investigated. Ileal chyme from piglets fed a diet containing different amounts of zinc oxide was used to evaluate a pyrosequencing study with barcoded 16S rRNA PCR products. Two DNA extraction methods (bead beating versus silica gel columns and two primer sets targeting variable regions of bacterial 16S rRNA genes (8f-534r versus 968f-1401r were considered. The SEED viewer software of the MG-RAST server was used for automated sequence analysis. A total of 5.2×105 sequences were used for analysis after processing for read length (150 bp, minimum sequence occurrence (5, and exclusion of eukaryotic and unclassified/uncultured sequences. DNA extraction procedures and primer sets differed significantly in total sequence yield. The distribution of bacterial order and main bacterial genera was influenced significantly by both parameters. However, this study has shown that the results of pyrosequencing studies using barcoded PCR amplicons of bacterial 16S rRNA genes depend on DNA extraction and primer choice, as well as on the manner of downstream sequence analysis.


    DEFF Research Database (Denmark)

    Pafilis, Evangelos; Buttigieg, Pier Luigi; Ferrell, Barbra


    The microbial and molecular ecology research communities have made substantial progress on developing standards for annotating samples with environment metadata. However, sample manual annotation is a highly labor intensive process and requires familiarity with the terminologies used. We have the...... and text-mining-assisted curation revealed that EXTRACT speeds up annotation by 15-25% and helps curators to detect terms that would otherwise have been missed.Database URL:

  5. EEG signal features extraction based on fractal dimension. (United States)

    Finotello, Francesca; Scarpa, Fabio; Zanon, Mattia


    The spread of electroencephalography (EEG) in countless applications has fostered the development of new techniques for extracting synthetic and informative features from EEG signals. However, the definition of an effective feature set depends on the specific problem to be addressed and is currently an active field of research. In this work, we investigated the application of features based on fractal dimension to a problem of sleep identification from EEG data. We demonstrated that features based on fractal dimension, including two novel indices defined in this work, add valuable information to standard EEG features and significantly improve sleep identification performance.

  6. Key frame extraction based on spatiotemporal motion trajectory (United States)

    Zhang, Yunzuo; Tao, Ran; Zhang, Feng


    Spatiotemporal motion trajectory can accurately reflect the changes of motion state. Motivated by this observation, this letter proposes a method for key frame extraction based on motion trajectory on the spatiotemporal slice. Different from the well-known motion related methods, the proposed method utilizes the inflexions of the motion trajectory on the spatiotemporal slice of all the moving objects. Experimental results show that although a similar performance is achieved in the single-objective screen, by comparing the proposed method to that achieved with the state-of-the-art methods based on motion energy or acceleration, the proposed method shows a better performance in a multiobjective video.

  7. An RNA-Seq-based reference transcriptome for Citrus. (United States)

    Terol, Javier; Tadeo, Francisco; Ventimilla, Daniel; Talon, Manuel


    Previous RNA-Seq studies in citrus have been focused on physiological processes relevant to fruit quality and productivity of the major species, especially sweet orange. Less attention has been paid to vegetative or reproductive tissues, while most Citrus species have never been analysed. In this work, we characterized the transcriptome of vegetative and reproductive tissues from 12 Citrus species from all main phylogenetic groups. Our aims were to acquire a complete view of the citrus transcriptome landscape, to improve previous functional annotations and to obtain genetic markers associated with genes of agronomic interest. 28 samples were used for RNA-Seq analysis, obtained from 12 Citrus species: C. medica, C. aurantifolia, C. limon, C. bergamia, C. clementina, C. deliciosa, C. reshni, C. maxima, C. paradisi, C. aurantium, C. sinensis and Poncirus trifoliata. Four different organs were analysed: root, phloem, leaf and flower. A total of 3421 million Illumina reads were produced and mapped against the reference C. clementina genome sequence. Transcript discovery pipeline revealed 3326 new genes, the number of genes with alternative splicing was increased to 19,739, and a total of 73,797 transcripts were identified. Differential expression studies between the four tissues showed that gene expression is overall related to the physiological function of the specific organs above any other variable. Variants discovery analysis revealed the presence of indels and SNPs in genes associated with fruit quality and productivity. Pivotal pathways in citrus such as those of flavonoids, flavonols, ethylene and auxin were also analysed in detail.

  8. Rare earth element enrichment using membrane based solvent extraction (United States)

    Makertiharta, I. G. B. N.; Dharmawijaya, P. T.; Zunita, M.; Wenten, I. G.


    The chemical, catalytic, electrical, magnetic, and optical properties of rare earth elements are required in broad applications. Rare earth elements have similar physical and chemical properties thus it is difficult to separate one from each other. Rare earth element is relatively abundant in earth's crust but rarely occur in high concentrated deposits. Traditionally, ion-exchange and solvent extraction techniques have been developed to separate and purify single rare earth solutions or compounds. Recently, membrane starts to gain attention for rare earth separation by combining membrane and proven technologies such as solvent extraction. Membrane-based process offers selective, reliable, energy efficient and easy to scale up separation. During membrane-based separation process, one phase passes through membrane pores while the other phase is rejected. There is no direct mixing of two phases thus the solvent loss is very low. Membrane can also lower solvent physical properties requirement (viscosity, density) and backmixing, eliminate flooding phenomenon and provide large interfacial area for mass transfer. This paper will summarize research efforts in developing membrane technology for rare earth element separation. Special attention will be given to solvent extraction related process as the commonly used method for rare earth element separation. Furthermore, membrane configuration and its potentials will also be discussed.

  9. Motion feature extraction scheme for content-based video retrieval (United States)

    Wu, Chuan; He, Yuwen; Zhao, Li; Zhong, Yuzhuo


    This paper proposes the extraction scheme of global motion and object trajectory in a video shot for content-based video retrieval. Motion is the key feature representing temporal information of videos. And it is more objective and consistent compared to other features such as color, texture, etc. Efficient motion feature extraction is an important step for content-based video retrieval. Some approaches have been taken to extract camera motion and motion activity in video sequences. When dealing with the problem of object tracking, algorithms are always proposed on the basis of known object region in the frames. In this paper, a whole picture of the motion information in the video shot has been achieved through analyzing motion of background and foreground respectively and automatically. 6-parameter affine model is utilized as the motion model of background motion, and a fast and robust global motion estimation algorithm is developed to estimate the parameters of the motion model. The object region is obtained by means of global motion compensation between two consecutive frames. Then the center of object region is calculated and tracked to get the object motion trajectory in the video sequence. Global motion and object trajectory are described with MPEG-7 parametric motion and motion trajectory descriptors and valid similar measures are defined for the two descriptors. Experimental results indicate that our proposed scheme is reliable and efficient.

  10. RNACompress: Grammar-based compression and informational complexity measurement of RNA secondary structure

    Directory of Open Access Journals (Sweden)

    Chen Chun


    Full Text Available Abstract Background With the rapid emergence of RNA databases and newly identified non-coding RNAs, an efficient compression algorithm for RNA sequence and structural information is needed for the storage and analysis of such data. Although several algorithms for compressing DNA sequences have been proposed, none of them are suitable for the compression of RNA sequences with their secondary structures simultaneously. This kind of compression not only facilitates the maintenance of RNA data, but also supplies a novel way to measure the informational complexity of RNA structural data, raising the possibility of studying the relationship between the functional activities of RNA structures and their complexities, as well as various structural properties of RNA based on compression. Results RNACompress employs an efficient grammar-based model to compress RNA sequences and their secondary structures. The main goals of this algorithm are two fold: (1 present a robust and effective way for RNA structural data compression; (2 design a suitable model to represent RNA secondary structure as well as derive the informational complexity of the structural data based on compression. Our extensive tests have shown that RNACompress achieves a universally better compression ratio compared with other sequence-specific or common text-specific compression algorithms, such as Gencompress, winrar and gzip. Moreover, a test of the activities of distinct GTP-binding RNAs (aptamers compared with their structural complexity shows that our defined informational complexity can be used to describe how complexity varies with activity. These results lead to an objective means of comparing the functional properties of heteropolymers from the information perspective. Conclusion A universal algorithm for the compression of RNA secondary structure as well as the evaluation of its informational complexity is discussed in this paper. We have developed RNACompress, as a useful tool

  11. Ground extraction from airborne laser data based on wavelet analysis (United States)

    Xu, Liang; Yang, Yan; Jiang, Bowen; Li, Jia


    With the advantages of high resolution and accuracy, airborne laser scanning data are widely used in topographic mapping. In order to generate a DTM, measurements from object features such as buildings, vehicles and vegetation have to be classified and removed. However, the automatic extraction of bare earth from point clouds acquired by airborne laser scanning equipment remains a problem in LIDAR data filtering nowadays. In this paper, a filter algorithm based on wavelet analysis is proposed. Relying on the capability of detecting discontinuities of continuous wavelet transform and the feature of multi-resolution analysis, the object points can be removed, while ground data are preserved. In order to evaluate the performance of this approach, we applied it to the data set used in the ISPRS filter test in 2003. 15 samples have been tested by the proposed approach. Results showed that it filtered most of the objects like vegetation and buildings, and extracted a well defined ground model.

  12. Modification of evidence theory based on feature extraction

    Institute of Scientific and Technical Information of China (English)

    DU Feng; SHI Wen-kang; DENG Yong


    Although evidence theory has been widely used in information fusion due to its effectiveness of uncertainty reasoning, the classical DS evidence theory involves counter-intuitive behaviors when high conflict information exists. Many modification methods have been developed which can be classified into the following two kinds of ideas, either modifying the combination rules or modifying the evidence sources. In order to make the modification more reasonable and more effective, this paper gives a thorough analysis of some typical existing modification methods firstly, and then extracts the intrinsic feature of the evidence sources by using evidence distance theory. Based on the extracted features, two modified plans of evidence theory according to the corresponding modification ideas have been proposed. The results of numerical examples prove the good performance of the plans when combining evidence sources with high conflict information.

  13. Features Extraction for Object Detection Based on Interest Point

    Directory of Open Access Journals (Sweden)

    Amin Mohamed Ahsan


    Full Text Available In computer vision, object detection is an essential process for further processes such as object tracking, analyzing and so on. In the same context, extraction features play important role to detect the object correctly. In this paper we present a method to extract local features based on interest point which is used to detect key-points within an image, then, compute histogram of gradient (HOG for the region surround that point. Proposed method used speed-up robust feature (SURF method as interest point detector and exclude the descriptor. The new descriptor is computed by using HOG method. The proposed method got advantages of both mentioned methods. To evaluate the proposed method, we used well-known dataset which is Caltech101. The initial result is encouraging in spite of using a small data for training.

  14. Music snippet extraction via melody-based repeated pattern discovery

    Institute of Scientific and Technical Information of China (English)

    XU JiePing; ZHAO Yang; CHEN Zhe; LIU ZiLi


    In this paper, we present a complete set of procedures to automatically extract a music snippet, defined as the most representative or the highlighted excerpt of a music clip. We first generate a modified and compact similarity matrix based on selected features and distance metrics, and then several improved techniques for music repeated pattern discovery are utilized because a music snippet is usually a part of the repeated melody, main theme or chorus. During the process, redundant and wrongly detected patterns are discarded, boundaries are corrected using beat information, and final clusters are also further sorted according to the occurrence frequency and energy information. Subsequently, following our methods, we designed a music snippet extraction system which allows users to detect snippets. Experiments performed on the system show the superiority of our proposed approach.

  15. Spindle extraction method for ISAR image based on Radon transform (United States)

    Wei, Xia; Zheng, Sheng; Zeng, Xiangyun; Zhu, Daoyuan; Xu, Gaogui


    In this paper, a method of spindle extraction of target in inverse synthetic aperture radar (ISAR) image is proposed which depends on Radon Transform. Firstly, utilizing Radon Transform to detect all straight lines which are collinear with these line segments in image. Then, using Sobel operator to detect image contour. Finally, finding all intersections of each straight line and image contour, the two intersections which have maximum distance between them is the two ends of this line segment and the longest line segment of all line segments is spindle of target. According to the proposed spindle extraction method, one hundred simulated ISAR images which are respectively rotated 0 degrees, 10 degrees, 20 degrees, 30 degrees and 40 degrees in counterclockwise are used to do experiment and the proposed method and the detection results are more close to the real spindle of target than the method based on Hough Transform .


    Directory of Open Access Journals (Sweden)

    Thiruvengatanadhan Ramalingam


    Full Text Available Audio classification serves as the fundamental step towards the rapid growth in audio data volume. Due to the increasing size of the multimedia sources speech and music classification is one of the most important issues for multimedia information retrieval. In this work a speech/music discrimination system is developed which utilizes the Discrete Wavelet Transform (DWT as the acoustic feature. Multi resolution analysis is the most significant statistical way to extract the features from the input signal and in this study, a method is deployed to model the extracted wavelet feature. Support Vector Machines (SVM are based on the principle of structural risk minimization. SVM is applied to classify audio into their classes namely speech and music, by learning from training data. Then the proposed method extends the application of Gaussian Mixture Models (GMM to estimate the probability density function using maximum likelihood decision methods. The system shows significant results with an accuracy of 94.5%.

  17. Location Fingerprint Extraction for Magnetic Field Magnitude Based Indoor Positioning

    Directory of Open Access Journals (Sweden)

    Wenhua Shao


    Full Text Available Smartphone based indoor positioning has greatly helped people in finding their positions in complex and unfamiliar buildings. One popular positioning method is by utilizing indoor magnetic field, because this feature is stable and infrastructure-free. In this method, the magnetometer embedded on the smartphone measures indoor magnetic field and queries its position. However, the environments of the magnetometer are rather harsh. This harshness mainly consists of coarse-grained hard/soft-iron calibrations and sensor electronic noise. The two kinds of interferences decrease the position distinguishability of the magnetic field. Therefore, it is important to extract location features from magnetic fields to reduce these interferences. This paper analyzes the main interference sources of the magnetometer embedded on the smartphone. In addition, we present a feature distinguishability measurement technique to evaluate the performance of different feature extraction methods. Experiments revealed that selected fingerprints will improve position distinguishability.

  18. Remote Sensing Image Feature Extracting Based Multiple Ant Colonies Cooperation

    Directory of Open Access Journals (Sweden)

    Zhang Zhi-long


    Full Text Available This paper presents a novel feature extraction method for remote sensing imagery based on the cooperation of multiple ant colonies. First, multiresolution expression of the input remote sensing imagery is created, and two different ant colonies are spread on different resolution images. The ant colony in the low-resolution image uses phase congruency as the inspiration information, whereas that in the high-resolution image uses gradient magnitude. The two ant colonies cooperate to detect features in the image by sharing the same pheromone matrix. Finally, the image features are extracted on the basis of the pheromone matrix threshold. Because a substantial amount of information in the input image is used as inspiration information of the ant colonies, the proposed method shows higher intelligence and acquires more complete and meaningful image features than those of other simple edge detectors.

  19. A molecular exploration of human DNA/RNA co-extracted from the palmar surface of the hands and fingers. (United States)

    Lacerenza, D; Aneli, S; Omedei, M; Gino, S; Pasino, S; Berchialla, P; Robino, C


    "Touch DNA" refers to the DNA that is left behind when a person touches or comes into contact with an item. However, the source of touch DNA is still debated and the large variability in DNA yield from casework samples suggests that, besides skin, various body fluids can be transferred through contact. Another important issue concerning touch DNA is the possible occurrence of secondary transfer, but the data published in the literature in relation to the background levels of foreign DNA present on the hand surfaces of the general population are very limited. As the present study aimed at better understanding the nature and characteristics of touch DNA, samples were collected from the palmar surface of the hands and fingers ("PHF" samples) of 30 male and 30 female donors by tape-lifting/swabbing and subjected to DNA/RNA co-extraction. Multiplex mRNA profiling showed that cellular material different from skin could be observed in 15% of the PHF samples. The total amount of DNA recovered from these samples (median 5.1 ng) was significantly higher than that obtained from samples containing skin cells only (median 1.6 ng). The integrity of the DNA isolated from the donors' hands and fingers as well as the prevalence of DNA mixtures were evaluated by STR typing and compared with reference STR profiles from buccal swabs. DNA integrity appeared significantly higher in the male rather than in the female subsample, as the average percentage of the donors' alleles effectively detected in PHF profiles was 75.1% and 60.1%, respectively. The prevalence of mixtures with a foreign DNA contribution ≥20% was 19.2% (30.0% in the female PHF samples and 8.3% in the male PHF samples). The obtained results support the hypothesis that transfer of cellular material different from skin may underlie the occasional recovery of quality STR profiles from handled items. These results also suggest that gender may represent an important factor influencing the propensity of individuals to carry

  20. Knowledge-based analysis of functional impacts of mutations in microRNA seed regions

    Indian Academy of Sciences (India)

    Anindya Bhattacharya; Yan Cui


    MicroRNAs are a class of important post-transcriptional regulators. Genetic and somatic mutations in miRNAs, especially those in the seed regions, have profound and broad impacts on gene expression and physiological and pathological processes. Over 500 SNPs were mapped to the miRNA seeds, which are located at position 2–8 of the mature miRNA sequences. We found that the central positions of the miRNA seeds contain fewer genetic variants and therefore are more evolutionary conserved than the peripheral positions in the seeds. We developed a knowledge-based method to analyse the functional impacts of mutations in miRNA seed regions. We computed the gene ontology-based similarity score GOSS and the GOSS percentile score for all 517 SNPs in miRNA seeds. In addition to the annotation of SNPs for their functional effects, in the present article we also present a detailed analysis pipeline for finding the key functional changes for seed SNPs. We performed a detailed gene ontology graph-based analysis of enriched functional categories for miRNA target gene sets. In the analysis of a SNP in the seed region of hsa-miR-96 we found that two key biological processes for progressive hearing loss `Neurotrophin TRK receptor signaling pathway' and `Epidermal growth factor receptor signaling pathway' were significantly and differentially enriched by the two sets of allele-specific target genes of miRNA hsa-miR-96.

  1. SiRNA sequence model: redesign algorithm based on available genome-wide libraries. (United States)

    Kozak, Karol


    The evolution of RNA interference (RNAi) and the development of technologies exploiting its biology have enabled scientists to rapidly examine the consequences of depleting a particular gene product in cells. Design tools have been developed based on experimental data to increase the knockdown efficiency of siRNAs. Not all siRNAs that are developed to a given target mRNA are equally effective. Currently available design algorithms take an accession, identify conserved regions among their transcript space, find accessible regions within the mRNA, design all possible siRNAs for these regions, filter them based on multi-scores thresholds, and then perform off-target filtration. These different criteria are used by commercial suppliers to produce siRNA genome-wide libraries for different organisms. In this article, we analyze existing siRNA design algorithms and evaluate weight of design parameters for libraries produced in the last decade. We proved that not all essential parameters are currently applied by siRNA vendors. Based on our evaluation results, we were able to suggest an siRNA sequence pattern. The findings in our study can be useful for commercial vendors improving the design of RNAi constructs, by addressing both the issue of potency and the issue of specificity.

  2. Nanotechnology-based strategies for the detection and quantification of microRNA. (United States)

    Degliangeli, Federica; Pompa, Pier Paolo; Fiammengo, Roberto


    MicroRNAs (miRNAs) are important regulators of gene expression, and many pathological conditions, including cancer, are characterized by altered miRNA expression levels. Therefore, accurate and sensitive quantification of miRNAs may result in correct disease diagnosis establishing these small noncoding RNA transcripts as valuable biomarkers. Aiming at overcoming some limitations of conventional quantification strategies, nanotechnology is currently providing numerous significant alternatives to miRNA sensing. In this review an up-to-date account of nanotechnology-based strategies for miRNA detection and quantification is given. The topics covered are: nanoparticle-based approaches in solution, sensing based on nanostructured surfaces, combined nanoparticle/surface sensing approaches, and single-molecule approaches.

  3. Base-pairing versatility determines wobble sites in tRNA anticodons of vertebrate mitogenomes.

    Directory of Open Access Journals (Sweden)

    Miguel M Fonseca

    Full Text Available BACKGROUND: Vertebrate mitochondrial genomes typically have one transfer RNA (tRNA for each synonymous codon family. This limited anticodon repertoire implies that each tRNA anticodon needs to wobble (establish a non-Watson-Crick base pairing between two nucleotides in RNA molecules to recognize one or more synonymous codons. Different hypotheses have been proposed to explain the factors that determine the nucleotide composition of wobble sites in vertebrate mitochondrial tRNA anticodons. Until now, the two major postulates--the "codon-anticodon adaptation hypothesis" and the "wobble versatility hypothesis"--have not been formally tested in vertebrate mitochondria because both make the same predictions regarding the composition of anticodon wobble sites. The same is true for the more recent "wobble cost hypothesis". PRINCIPAL FINDINGS: In this study we have analyzed the occurrence of synonymous codons and tRNA anticodon wobble sites in 1553 complete vertebrate mitochondrial genomes, focusing on three fish species with mtDNA codon usage bias reversal (L-strand is GT-rich. These mitogenomes constitute an excellent opportunity to study the evolution of the wobble nucleotide composition of tRNA anticodons because due to the reversal the predictions for the anticodon wobble sites differ between the existing hypotheses. We observed that none of the wobble sites of tRNA anticodons in these unusual mitochondrial genomes coevolved to match the new overall codon usage bias, suggesting that nucleotides at the wobble sites of tRNA anticodons in vertebrate mitochondrial genomes are determined by wobble versatility. CONCLUSIONS/SIGNIFICANCE: Our results suggest that, at wobble sites of tRNA anticodons in vertebrate mitogenomes, selection favors the most versatile nucleotide in terms of wobble base-pairing stability and that wobble site composition is not influenced by codon usage. These results are in agreement with the "wobble versatility hypothesis".

  4. The ReNAissanCe of mRNA-based cancer therapy. (United States)

    Van Lint, Sandra; Renmans, Dries; Broos, Katrijn; Dewitte, Heleen; Lentacker, Ine; Heirman, Carlo; Breckpot, Karine; Thielemans, Kris


    About 25 years ago, mRNA became a tool of interest in anticancer vaccination approaches. However, due to its rapid degradation in situ, direct application of mRNA was confronted with considerable skepticism during its early use. Consequently, mRNA was for a long time mainly used for the ex vivo transfection of dendritic cells, professional antigen-presenting cells known to stimulate immunity. The interest in direct application of mRNA experienced a revival, as researchers became aware of the many advantages mRNA offers. Today, mRNA is considered to be an ideal vehicle for the induction of strong immune responses against cancer. The growing numbers of preclinical trials and as a consequence the increasing clinical application of mRNA as an off-the-shelf anticancer vaccine signifies a renaissance for transcript-based antitumor therapy. In this review, we highlight this renaissance using a timeline providing all milestones in the application of mRNA for anticancer vaccination.

  5. Next-generation libraries for robust RNA interference-based genome-wide screens. (United States)

    Kampmann, Martin; Horlbeck, Max A; Chen, Yuwen; Tsai, Jordan C; Bassik, Michael C; Gilbert, Luke A; Villalta, Jacqueline E; Kwon, S Chul; Chang, Hyeshik; Kim, V Narry; Weissman, Jonathan S


    Genetic screening based on loss-of-function phenotypes is a powerful discovery tool in biology. Although the recent development of clustered regularly interspaced short palindromic repeats (CRISPR)-based screening approaches in mammalian cell culture has enormous potential, RNA interference (RNAi)-based screening remains the method of choice in several biological contexts. We previously demonstrated that ultracomplex pooled short-hairpin RNA (shRNA) libraries can largely overcome the problem of RNAi off-target effects in genome-wide screens. Here, we systematically optimize several aspects of our shRNA library, including the promoter and microRNA context for shRNA expression, selection of guide strands, and features relevant for postscreen sample preparation for deep sequencing. We present next-generation high-complexity libraries targeting human and mouse protein-coding genes, which we grouped into 12 sublibraries based on biological function. A pilot screen suggests that our next-generation RNAi library performs comparably to current CRISPR interference (CRISPRi)-based approaches and can yield complementary results with high sensitivity and high specificity.

  6. A collaborative European exercise on mRNA-based body fluid/skin typing and interpretation of DNA and RNA results

    DEFF Research Database (Denmark)

    van den Berge, M; Carracedo, A; Gomes, I;


    The European Forensic Genetics Network of Excellence (EUROFORGEN-NoE) undertook a collaborative project on mRNA-based body fluid/skin typing and the interpretation of the resulting RNA and DNA data. Although both body fluids and skin are composed of a variety of cell types with different function...

  7. Mechanism of gold solvent extraction from aurocyanide solution by quaternary amines: models of extracting species based on hydrogen bonding

    Institute of Scientific and Technical Information of China (English)


    The mechanism of gold solvent extraction from KAu(CN)2 solution was investigated by means of FTIR, EXAFS, ICP and radioactive tracer methods. Two extraction systems were studied, namely N263-tributyl phosphate(TBP)-n-dodecane and N263-iso-octanol-n-dodecane. High-reso- lution FT IR spectroscopy indicated that the CN stretching vibrations of the two extraction systems differred greatly. In order to interpret the significant difference in CN stretching vibrations, two extracting species models are proposed supramolecular structures based on the formation of hydrogen bonds between Au(CN)2- and modifiers such as TBP and iso-octanol.

  8. Comparative analysis of eukaryotic marine microbial assemblages from 18S rRNA gene and gene transcript clone libraries by using different methods of extraction. (United States)

    Koid, Amy; Nelson, William C; Mraz, Amy; Heidelberg, Karla B


    Eukaryotic marine microbes play pivotal roles in biogeochemical nutrient cycling and ecosystem function, but studies that focus on the protistan biogeography and genetic diversity lag-behind studies of other microbes. 18S rRNA PCR amplification and clone library sequencing are commonly used to assess diversity that is culture independent. However, molecular methods are not without potential biases and artifacts. In this study, we compare the community composition of clone libraries generated from the same water sample collected at the San Pedro Ocean Time Series (SPOTs) station in the northwest Pacific Ocean. Community composition was assessed using different cell lysis methods (chemical and mechanical) and the extraction of different nucleic acids (DNA and RNA reverse transcribed to cDNA) to build Sanger ABI clone libraries. We describe specific biases for ecologically important phylogenetic groups resulting from differences in nucleic acid extraction methods that will inform future designs of eukaryotic diversity studies, regardless of the target sequencing platform planned.

  9. Antioxidant Activity and Induction of mRNA Expressions of Antioxidant Enzymes in HEK-293 Cells of Moringa oleifera Leaf Extract. (United States)

    Vongsak, Boonyadist; Mangmool, Supachoke; Gritsanapan, Wandee


    The leaves of Moringa oleifera, collected in different provinces in Thailand, were determined for the contents of total phenolics, total flavonoids, major components, and antioxidant activity. The extract and its major active components were investigated for the inhibition of H2O2-induced reactive oxygen species production and the effects on antioxidant enzymes mRNA expression. The extract, crypto-chlorogenic acid, isoquercetin and astragalin, significantly reduced the reactive oxygen species production inducing by H2O2 in HEK-293 cells. Treatment with isoquercetin significantly increased the mRNA expression levels of antioxidant enzymes such as superoxide dismutase, catalase and heme oxygenase 1. These results confirm that M. oleifera leaves are good sources of natural antioxidant with isoquercetin as an active compound.

  10. Leaf Vein Extraction Based on Gray-scale Morphology

    Directory of Open Access Journals (Sweden)

    Xiaodong Zheng


    Full Text Available Leaf features play an important role in plant species identification and plant taxonomy. The type of the leaf vein is an important morphological feature of the leaf in botany. Leaf vein should be extracted from the leaf in the image before discriminating its type. In this paper a new method of leaf vein extraction has been proposed based on gray-scale morphology. Firstly, the color image of the plant leaf is transformed to the gray image according to the hue and intensity information. Secondly, the gray-scale morphology processing is applied to the image to eliminate the color overlap in the whole leaf vein and the whole background. Thirdly, the linear intensity adjustment is adopted to enlarge the gray value difference between the leaf vein and its background. Fourthly, calculate a threshold with OSTU method to segment the leaf vein from its background. Finally, the leaf vein can be got after some processing on details. Experiments have been conducted with several images. The results show the effectiveness of the method. The idea of the method is also applicable to other linear objects extraction.

  11. Analogy between gambling and measurement-based work extraction (United States)

    Vinkler, Dror A.; Permuter, Haim H.; Merhav, Neri


    In information theory, one area of interest is gambling, where mutual information characterizes the maximal gain in wealth growth rate due to knowledge of side information; the betting strategy that achieves this maximum is named the Kelly strategy. In the field of physics, it was recently shown that mutual information can characterize the maximal amount of work that can be extracted from a single heat bath using measurement-based control protocols, i.e. using ‘information engines’. However, to the best of our knowledge, no relation between gambling and information engines has been presented before. In this paper, we briefly review the two concepts and then demonstrate an analogy between gambling, where bits are converted into wealth, and information engines, where bits representing measurements are converted into energy. From this analogy follows an extension of gambling to the continuous-valued case, which is shown to be useful for investments in currency exchange rates or in the stock market using options. Moreover, the analogy enables us to use well-known methods and results from one field to solve problems in the other. We present three such cases: maximum work extraction when the probability distributions governing the system and measurements are unknown, work extraction when some energy is lost in each cycle, e.g. due to friction, and an analysis of systems with memory. In all three cases, the analogy enables us to use known results in order to obtain new ones.

  12. Optimal consistency in microRNA expression analysis using reference-gene-based normalization. (United States)

    Wang, Xi; Gardiner, Erin J; Cairns, Murray J


    Normalization of high-throughput molecular expression profiles secures differential expression analysis between samples of different phenotypes or biological conditions, and facilitates comparison between experimental batches. While the same general principles apply to microRNA (miRNA) normalization, there is mounting evidence that global shifts in their expression patterns occur in specific circumstances, which pose a challenge for normalizing miRNA expression data. As an alternative to global normalization, which has the propensity to flatten large trends, normalization against constitutively expressed reference genes presents an advantage through their relative independence. Here we investigated the performance of reference-gene-based (RGB) normalization for differential miRNA expression analysis of microarray expression data, and compared the results with other normalization methods, including: quantile, variance stabilization, robust spline, simple scaling, rank invariant, and Loess regression. The comparative analyses were executed using miRNA expression in tissue samples derived from subjects with schizophrenia and non-psychiatric controls. We proposed a consistency criterion for evaluating methods by examining the overlapping of differentially expressed miRNAs detected using different partitions of the whole data. Based on this criterion, we found that RGB normalization generally outperformed global normalization methods. Thus we recommend the application of RGB normalization for miRNA expression data sets, and believe that this will yield a more consistent and useful readout of differentially expressed miRNAs, particularly in biological conditions characterized by large shifts in miRNA expression.

  13. Mapping Optimal Charge Density and Length of ROMP-Based PTDMs for siRNA Internalization. (United States)

    Caffrey, Leah M; deRonde, Brittany M; Minter, Lisa M; Tew, Gregory N


    A fundamental understanding of how polymer structure impacts internalization and delivery of biologically relevant cargoes, particularly small interfering ribonucleic acid (siRNA), is of critical importance to the successful design of improved delivery reagents. Herein we report the use of ring-opening metathesis polymerization (ROMP) methods to synthesize two series of guanidinium-rich protein transduction domain mimics (PTDMs): one based on an imide scaffold that contains one guanidinium moiety per repeat unit, and another based on a diester scaffold that contains two guanidinium moieties per repeat unit. By varying both the degree of polymerization and, in effect, the relative number of cationic charges in each PTDM, the performances of the two ROMP backbones for siRNA internalization were evaluated and compared. Internalization of fluorescently labeled siRNA into Jurkat T cells demonstrated that fluorescein isothiocyanate (FITC)-siRNA internalization had a charge content dependence, with PTDMs containing approximately 40 to 60 cationic charges facilitating the most internalization. Despite this charge content dependence, the imide scaffold yielded much lower viabilities in Jurkat T cells than the corresponding diester PTDMs with similar numbers of cationic charges, suggesting that the diester scaffold is preferred for siRNA internalization and delivery applications. These developments will not only improve our understanding of the structural factors necessary for optimal siRNA internalization, but will also guide the future development of optimized PTDMs for siRNA internalization and delivery.

  14. 油茶总RNA的提取与内参基因的初选%RNA extraction and primary screening of reference genes in Camellia oleifera

    Institute of Scientific and Technical Information of China (English)

    宋志波; 刘敏; 贾宝光; 田雷; 曾艳玲; 周俊琴; 谭晓风; 张琳


    为得到高质量的油茶总RNA及稳定的内参基因,以油茶良种‘华硕’为试验材料,采用ambion试剂盒结合CTAB法进行总RNA提取。获得的总RNA完整性好,纯度高;从前期构建的油茶转录组数据中选取ACT(肌动蛋白基因)等14个基因作为候选内参基因,共设计22对引物,通过RT-PCR反应体系的优化及琼脂糖凝胶电泳检测,快速筛选出了适合油茶不同组织(器官)的内参基因。ALB(清蛋白基因),EF1α(延伸因子基因),ETIF3H(启动因子基因),UBC2(泛素结合酶基因)可以作为油茶果实初选内参基因,EF1α可以作为油茶根、茎、花及果实内参基因,ETIF3H可以作为油茶根、茎、叶、花瓣的内参基因。%In order to obtain high quality total RNA and stable reference genes in Camellia oleifera, taking ifne cultivar‘Huashuo’ as materials, total RNA was extracted by a combination of ambion kit and CTAB method. The obtained total RNA has good integrity and high purity. A total of 14 genes were selected as candidates of reference genes based on the previously constructed transcriptome data in C. oleifera, and 22 primers were designed for the 14 genes. The reference genes suitable for different tissues or organs were rapidly screened out by RT-PCR reaction system optimization and agarose gel electrophoresis detection. ALB (albumin gene), EF1α (elongation factor gene), ETIF3H (eukaryotic translation initiation gene), UBC2 (ubiquitin-conjugating enzyme gene) can be used as the reference genes for fruits, EF1αcan be used as a reference gene for root, stem, lfower and fruit, and ETIF3H can be used as a reliable reference gene for root, stem, leaf and fruit in C. oleifera.

  15. Biosensor-based microRNA detection: techniques, design, performance, and challenges. (United States)

    Johnson, Blake N; Mutharasan, Raj


    The current state of biosensor-based techniques for amplification-free microRNA (miRNA) detection is critically reviewed. Comparison with non-sensor and amplification-based molecular techniques (MTs), such as polymerase-based methods, is made in terms of transduction mechanism, associated protocol, and sensitivity. Challenges associated with miRNA hybridization thermodynamics which affect assay selectivity and amplification bias are briefly discussed. Electrochemical, electromechanical, and optical classes of miRNA biosensors are reviewed in terms of transduction mechanism, limit of detection (LOD), time-to-results (TTR), multiplexing potential, and measurement robustness. Current trends suggest that biosensor-based techniques (BTs) for miRNA assay will complement MTs due to the advantages of amplification-free detection, LOD being femtomolar (fM)-attomolar (aM), short TTR, multiplexing capability, and minimal sample preparation requirement. Areas of future importance in miRNA BT development are presented which include focus on achieving high measurement confidence and multiplexing capabilities.

  16. Multispectral Image Road Extraction Based Upon Automated Map Conflation (United States)

    Chen, Bin

    Road network extraction from remotely sensed imagery enables many important and diverse applications such as vehicle tracking, drone navigation, and intelligent transportation studies. There are, however, a number of challenges to road detection from an image. Road pavement material, width, direction, and topology vary across a scene. Complete or partial occlusions caused by nearby buildings, trees, and the shadows cast by them, make maintaining road connectivity difficult. The problems posed by occlusions are exacerbated with the increasing use of oblique imagery from aerial and satellite platforms. Further, common objects such as rooftops and parking lots are made of materials similar or identical to road pavements. This problem of common materials is a classic case of a single land cover material existing for different land use scenarios. This work addresses these problems in road extraction from geo-referenced imagery by leveraging the OpenStreetMap digital road map to guide image-based road extraction. The crowd-sourced cartography has the advantages of worldwide coverage that is constantly updated. The derived road vectors follow only roads and so can serve to guide image-based road extraction with minimal confusion from occlusions and changes in road material. On the other hand, the vector road map has no information on road widths and misalignments between the vector map and the geo-referenced image are small but nonsystematic. Properly correcting misalignment between two geospatial datasets, also known as map conflation, is an essential step. A generic framework requiring minimal human intervention is described for multispectral image road extraction and automatic road map conflation. The approach relies on the road feature generation of a binary mask and a corresponding curvilinear image. A method for generating the binary road mask from the image by applying a spectral measure is presented. The spectral measure, called anisotropy-tunable distance (ATD

  17. Activated charcoal-mediated RNA extraction method for Azadirachta indica and plants highly rich in polyphenolics, polysaccharides and other complex secondary compounds


    Rajakani, Raja; Narnoliya, Lokesh; Sangwan, Neelam Singh; Sangwan, Rajender Singh; Gupta, Vikrant


    Background High quality RNA is a primary requisite for numerous molecular biological applications but is difficult to isolate from several plants rich in polysaccharides, polyphenolics and other secondary metabolites. These compounds either bind with nucleic acids or often co-precipitate at the final step and many times cannot be removed by conventional methods and kits. Addition of vinyl-pyrollidone polymers in extraction buffer efficiently removes polyphenolics to some extent, but, it faile...

  18. Phase shift extraction algorithm based on Euclidean matrix norm. (United States)

    Deng, Jian; Wang, Hankun; Zhang, Desi; Zhong, Liyun; Fan, Jinping; Lu, Xiaoxu


    In this Letter, the character of Euclidean matrix norm (EMN) of the intensity difference between phase-shifting interferograms, which changes in sinusoidal form with the phase shifts, is presented. Based on this character, an EMN phase shift extraction algorithm is proposed. Both the simulation calculation and experimental research show that the phase shifts with high precision can be determined with the proposed EMN algorithm easily. Importantly, the proposed EMN algorithm will supply a powerful tool for the rapid calibration of the phase shifts.

  19. Automatic Foreground Extraction Based on Difference of Gaussian

    Directory of Open Access Journals (Sweden)

    Yubo Yuan


    Full Text Available A novel algorithm for automatic foreground extraction based on difference of Gaussian (DoG is presented. In our algorithm, DoG is employed to find the candidate keypoints of an input image in different color layers. Then, a keypoints filter algorithm is proposed to get the keypoints by removing the pseudo-keypoints and rebuilding the important keypoints. Finally, Normalized cut (Ncut is used to segment an image into several regions and locate the foreground with the number of keypoints in each region. Experiments on the given image data set demonstrate the effectiveness of our algorithm.

  20. Kernel-Based Learning for Domain-Specific Relation Extraction (United States)

    Basili, Roberto; Giannone, Cristina; Del Vescovo, Chiara; Moschitti, Alessandro; Naggar, Paolo

    In a specific process of business intelligence, i.e. investigation on organized crime, empirical language processing technologies can play a crucial role. The analysis of transcriptions on investigative activities, such as police interrogatories, for the recognition and storage of complex relations among people and locations is a very difficult and time consuming task, ultimately based on pools of experts. We discuss here an inductive relation extraction platform that opens the way to much cheaper and consistent workflows. The presented empirical investigation shows that accurate results, comparable to the expert teams, can be achieved, and parametrization allows to fine tune the system behavior for fitting domain-specific requirements.

  1. Probabilistic contour extraction based on shape prior model

    Institute of Scientific and Technical Information of China (English)

    FAN Xin; LIANG De-qun


    Statistical shape prior model is employed to construct the dynamics in probabilistic contour estimation.By applying principal component analysis,plausible shape samples are efficiently generated to predict contour samples.Based on the shape-dependent dynamics and probabilistic image model,a particle filter is used to estimate the contour with a specific shape.Compared with the deterministic approach with shape information,the proposed method is simple yet more effective in extracting contours from images with shape variations and occlusion.


    Directory of Open Access Journals (Sweden)



    Full Text Available Recent developments in digital video and drastic increase of internet use have increased the amount of people searching and watching videos online. In order to make the search of the videos easy, Summary of the video may be provided along with each video. The video summary provided thus should be effective so that the user would come to know the content of the video without having to watch it fully. The summary produced should consists of the key frames that effectively express the content and context of the video. This work suggests a method to extract key frames which express most of the information in the video. This is achieved by quantifying Visual attention each frame commands. Visual attention of each frame is quantified using a descriptor called Attention quantifier. This quantification of visual attention is based on the human attention mechanism that indicates color conspicuousness and the motion involved seek more attention. So based on the color conspicuousness and the motion involved each frame is given a Attention parameter. Based on the attention quantifier value the key frames are extracted and are summarized adaptively. This framework suggests a method to produces meaningful video summary.

  3. Evaluation of RNA extraction methods and identification of putative reference genes for real-time quantitative polymerase chain reaction expression studies on olive (Olea europaea L.) fruits. (United States)

    Nonis, Alberto; Vezzaro, Alice; Ruperti, Benedetto


    Genome wide transcriptomic surveys together with targeted molecular studies are uncovering an ever increasing number of differentially expressed genes in relation to agriculturally relevant processes in olive (Olea europaea L). These data need to be supported by quantitative approaches enabling the precise estimation of transcript abundance. qPCR being the most widely adopted technique for mRNA quantification, preliminary work needs to be done to set up robust methods for extraction of fully functional RNA and for the identification of the best reference genes to obtain reliable quantification of transcripts. In this work, we have assessed different methods for their suitability for RNA extraction from olive fruits and leaves and we have evaluated thirteen potential candidate reference genes on 21 RNA samples belonging to fruit developmental/ripening series and to leaves subjected to wounding. By using two different algorithms, GAPDH2 and PP2A1 were identified as the best reference genes for olive fruit development and ripening, and their effectiveness for normalization of expression of two ripening marker genes was demonstrated.

  4. Real-time PCR analysis of RNA extracted from formalin-fixed and paraffin-embeded tissues: effects of the fixation on outcome reliability. (United States)

    Castiglione, Francesca; Rossi Degl'Innocenti, Duccio; Taddei, Antonio; Garbini, Francesca; Buccoliero, Anna Maria; Raspollini, Maria Rosaria; Pepi, Monica; Paglierani, Milena; Asirelli, Grazia; Freschi, Giancarlo; Bechi, Paolo; Taddei, Gian Luigi


    In many pathologic circumstances, quantitative mRNA expression levels are important for evaluation of possible genome mutations. The development of real-time polymerase chain reaction (RT-PCR) technology has facilitated the realization of nucleic acid quantification. Potentially, quantitative PCR offers a number of advantages over traditional methods because it permits the use of small amounts of genetic material. In the present study, we optimize a RNA purification technique on specimens that are formalin-fixed, paraffin-embedded and we examine prolonged formalin fixation effects on quantitative RT-PCR analysis. We compared RNA levels with 70 colic mucosa samples using the cyclooxygenase 2 gene as marker. The difference in amplification successes between formalin-fixed tissues and formalin-fixed, paraffin-embedded tissues was not statistically significant. Moreover, we compared the expression of formalin-fixed samples with the expression of each fresh tissue. Wilcoxon Mann-Whitney test shows that only the difference in the expression levels of 1- or 3-hour formalin-fixed samples is not statistically significant with respect to other fixation times. We found that the mRNA can be reliably extracted from formalin fixed, paraffin-embedded tissue sections but that prolonged formalin fixation produces different results in quantitative RT-PCR. It can be related to difference in RNA sequences length and the generation of secondary structures that are more susceptible to the prolonged formalin fixation. We suppose that the paraffin do not influence the RNA extraction yield because there are no statistical significant differences between amplification success of formalin-fixed tissues and paraffin-embedded tissues. Therefore, in relative expression quantization, we confirm that it is appropriate to use specimens with same protocols and time for formalin fixation.

  5. Study on Total RNA Extraction Methods from Miracle Fruit%神秘果果实总RNA提取方法比较研究

    Institute of Scientific and Technical Information of China (English)

    谭才邓; 李静; 邓毛程


      以神秘果果实为材料,以获得高质量RNA为目的,以针对性地去除多糖、酚类物质为出发点,比较了Trizol法、CTAB法,并相应加以改良。结果显示:CTAB粗提法方便快捷,CTAB精提法提取的总RNA完整性好、纯度高,RT-PCR反应表明提取的总RNA都能满足分子生物学实验的要求。%  In order to extract total RNA form miracle fruit and reduce the influence of polysaccharides and polyphenolics, we studied and compared four RNA extraction methods such as Trizol method, Trizol improved method, CTAB crude method and CTAB refined method. The results showed that: The CTAB crude extraction method was convenient and quick, total RAN from the CTAB refined method was proved to be relatively intact and high purity, RT-PCR analysis showed that the total RNA from both CTAB methods could be used in further molecular biology research.

  6. Stealth monoolein-based nanocarriers for delivery of siRNA to cancer cells


    Oliveira, Ana Cristina Norberto Gonçalves; Raemdonck, Koen; Martens, Thomas; Rombouts, Koen; Simón-Vázquez, Rosana; Botelho, C.M.; Lopes, Ivo Edgar Araújo; Lúcio, M.; González-Fernández, África; Real Oliveira, M. Elisabete C.D.; Gomes, Andreia; Braeckmans, Kevin


    While the delivery of small interfering RNAs (siRNAs) is an attractive strategy to treat several clinical con- ditions, siRNA-nanocarriers stability after intravenous administration is still a major obstacle for the development of RNA-interference based therapies. But, although the need for stability is well recognized, the notion that strong stabilization can decrease nanocarriers efficiency is sometimes neglected. In this work we evaluated two stealth functionalization strategies to stabili...

  7. 一种自制的RNA分离试剂及其在鱼组织总RNA提取中的应用%A homemade RNA isolation reagent and its application in the extraction of fish tissue total RNA

    Institute of Scientific and Technical Information of China (English)

    徐坤; 李铎; 张优优; 李陇平; 麻丽霞; 张智英


    【Objective】 The study was done to test a homemade RNA isolation reagent,which is convenient and economical.【Method】 With TRizol Reagent as a comparison,the homemade RNA isolation reagent was used to extract total RNA of grass carp derma.The quality of the total RNA was checked with a spectrophotometer,denaturing agarose gel electrophoresis and also RT-PCR analysis.Finally,the total RNA was used to synthesize grass carp derma cDNA.Subsequently,we applied the homemade reagent to extract total RNA of zebra fish,which was used for RT-PCR to clone the full length zebra fish E2F5 gene(1 092 bp).Then an eukaryotic expression vector pcDNA3.1-flag-Zf E2F5 and a yeast expression vector pGAD GH-Zf E2F5 were constructed,and the cloned E2F5 gene was sequenced.【Result】 The total RNA of grass carp derma extracted with the homemade RNA isolation reagent was highly qualified as that with TRizol Reagent to synthesize grass carp cDNA.The total RNA of zebra fish extracted with the reagent got a even higher quality,with which the full length E2F5 gene was successfully cloned to construct pcDNA3.1-flag-Zf E2F5 and pGAD GH-Zf E2F5.【Conclusion】 The homemade RNA isolation reagent is suitable for the extraction of fish tissue total RNA,which will satisfy the needs of cDNA synthesis and gene cloning by RT-PCR.With the total RNA extracted using the homemade reagent,we successfully conducted the grass carp cDNA synthesis and zebra fish E2F5 cloning.These will be essential materials for our future studies.%【目的】尝试自制一种价格低廉的RNA分离试剂,并检验其应用效果。【方法】以商业化的TRizolReagent为对照,使用自制RNA分离试剂提取草鱼皮肤组织总RNA,采用分光光度计法、变性琼脂糖凝胶电泳法和RT-PCR检测其品质,并用提取的总RNA合成草鱼皮肤cDNA。同时使用自制RNA分离试剂提取斑马鱼总RNA,通过RT-PCR进行E2F5基因全长(1 092bp)的克隆,构建其真核和酵母表达载体并

  8. Maintaining Breast Cancer Specimen Integrity and Individual or Simultaneous Extraction of Quality DNA, RNA, and Proteins from Allprotect-Stabilized and Nonstabilized Tissue Samples

    LENUS (Irish Health Repository)

    Mee, Blanaid C.


    The Saint James\\'s Hospital Biobank was established in 2008, to develop a high-quality breast tissue BioResource, as a part of the breast cancer clinical care pathway. The aims of this work were: (1) to ascertain the quality of RNA, DNA, and protein in biobanked carcinomas and normal breast tissues, (2) to assess the efficacy of AllPrep® (Qiagen) in isolating RNA, DNA, and protein simultaneously, (3) to compare AllPrep with RNEasy® and QIAamp® (both Qiagen), and (4) to examine the effectiveness of Allprotect® (Qiagen), a new tissue stabilization medium in preserving DNA, RNA, and proteins. One hundred eleven frozen samples of carcinoma and normal breast tissue were analyzed. Tumor and normal tissue morphology were confirmed by frozen sections. Tissue type, tissue treatment (Allprotect vs. no Allprotect), extraction kit, and nucleic acid quantification were analyzed by utilizing a 4 factorial design (SPSS PASW 18 Statistics Software®). QIAamp (DNA isolation), AllPrep (DNA, RNA, and Protein isolation), and RNeasy (RNA isolation) kits were assessed and compared. Mean DNA yield and A260\\/280 values using QIAamp were 33.2 ng\\/μL and 1.86, respectively, and using AllPrep were 23.2 ng\\/μL and 1.94. Mean RNA yield and RNA Integrity Number (RIN) values with RNeasy were 73.4 ng\\/μL and 8.16, respectively, and with AllPrep were 74.8 ng\\/μL and 7.92. Allprotect-treated tissues produced higher RIN values of borderline significance (P=0.055). No discernible loss of RNA stability was detected after 6 h incubation of stabilized or nonstabilized tissues at room temperature or 4°C or in 9 freeze-thaw cycles. Allprotect requires further detailed evaluation, but we consider AllPrep to be an excellent option for the simultaneous extraction of RNA, DNA, and protein from tumor and normal breast tissues. The essential presampling procedures that maintain the diagnostic integrity of pathology specimens do not appear to compromise the quality of molecular isolates.

  9. An improved method for RNA extraction from urediniospores of and wheat leaves infected by an obligate fungal pathogen, Puccinia striiformis f. sp.tritici

    Institute of Scientific and Technical Information of China (English)

    MA Li-Jie; QIAO Jia-xing; KONG Xin-yu; WANG Jun-juan; XU Xiang-ming; HU Xiao-ping


    Stripe rust, caused byPuccinia striformis f. sp.tritici, is an important wheat disease in China, seriously threatening wheat production. Understanding the winter survival of the fungus is a key for predicting the spring epidemics of the disease, which determines the crop loss. Estimation ofP. striformisf. sp. triticiwinter survival requires processing a large number of samples for sensitive detection of the pathogen in wheat leaf tissue using real-time quantitative reverse transcription PCR (qRT-PCR). A bottleneck for the analysis is the acquisition of a good yield of high quality RNA suitable for qRT-PCR to distinguish dead and alive fungal hyphae inside leaves. Although several methods have been described in the literatures and commercial kits are available for RNA extraction, these methods are mostly too complicated, expensive and inefifcient. Thus, we modiifed three previously reported RNA extraction methods with common and low-cost reagents (LiCl, SDS and NaCl) to solve the problems and selected the best to obtain high quality and quantity RNA for use in qRT-PCR. In the three improved methods, the NaCl method was proven to be the best for extracting RNA from urediniospores of and wheat leaves infected byP. striformisf. sp. tritici, although the modiifed LiCl and SDS methods also increased yield of RNA compared to the previous methods. The improved NaCl method has the folowing advantages: 1) Complete transfer of urediniospores ofP. striformis f. sp. tritici from the mortar and pestle can ensure the initial amount of RNA for the qRT-PCR analysis; 2) the use of low-cost NaCl to replace more expensive Trizol can reduce the cost; 3) the yield and quality of RNA can be increased;4) the improved method is more suitable for a large number and high quantity of samples from ifelds. Using the improved NaCl method, the amount of RNA was increased three times from urediniospores ofP. striformis f. sp. triticicompared from the extraction kit. Approximately, 10.11 μg total

  10. Prediction of MicroRNA-Disease Associations Based on Social Network Analysis Methods

    Directory of Open Access Journals (Sweden)

    Quan Zou


    Full Text Available MicroRNAs constitute an important class of noncoding, single-stranded, ~22 nucleotide long RNA molecules encoded by endogenous genes. They play an important role in regulating gene transcription and the regulation of normal development. MicroRNAs can be associated with disease; however, only a few microRNA-disease associations have been confirmed by traditional experimental approaches. We introduce two methods to predict microRNA-disease association. The first method, KATZ, focuses on integrating the social network analysis method with machine learning and is based on networks derived from known microRNA-disease associations, disease-disease associations, and microRNA-microRNA associations. The other method, CATAPULT, is a supervised machine learning method. We applied the two methods to 242 known microRNA-disease associations and evaluated their performance using leave-one-out cross-validation and 3-fold cross-validation. Experiments proved that our methods outperformed the state-of-the-art methods.

  11. Multifunctional nanocarrier based on clay nanotubes for efficient intracellular siRNA delivery and gene silencing. (United States)

    Wu, Hui; Shi, Yinfeng; Huang, Chusen; Zhang, Yang; Wu, Jiahui; Shen, Hebai; Jia, Nengqin


    RNA interference-mediated gene silencing relating to disease has recently emerged as a powerful method in gene therapy. Despite the promises, effective transport of siRNA with minimal side effects remains a challenge. Halloysites are cheap and naturally available aluminosilicate clay nanotubes with high mechanical strength and biocompatibility. In this study, a novel multifunctional nanocarrier based on functionalized halloysite nanotubes (f-HNTs) has been developed via electrostatic layer-by-layer assembling approach for loading and intracellular delivery of therapeutic antisurvivin siRNA and simultaneously tracking their intracellular transport, in which PEI-modified HNTs are used as gene vector, antisurvivin siRNA as gene therapeutic agent, and mercaptoacetic acid-capped CdSe quantum dots as fluorescent labeling probes. The successful assembly of the f-HNTs-siRNA complexes was systematically characterized by transmission electron microscopy (TEM), UV-visible spectrophotometry, Zeta potential measurement, fluorescence spectrophotometry, and electrochemical impedance spectroscopy. Confocal microscopy, biological TEM, and flow cytometry studies revealed that the complexes enabled the efficient intracellular delivery of siRNA for cell-specific gene silencing. MTT assays exhibited that the complexes can enhance antitumor activity. Furthermore, Western blot analysis showed that f-HNTs-mediated siRNA delivery effectively knocked down gene expression of survivin and thereby decreased the levels of target proteins of PANC-1 cells. Therefore, this study suggested that the synthesized f-HNTs were a new effective drug delivery system for potential application in cancer gene therapy.

  12. A Meta-Path-Based Prediction Method for Human miRNA-Target Association

    Directory of Open Access Journals (Sweden)

    Jiawei Luo


    Full Text Available MicroRNAs (miRNAs are short noncoding RNAs that play important roles in regulating gene expressing, and the perturbed miRNAs are often associated with development and tumorigenesis as they have effects on their target mRNA. Predicting potential miRNA-target associations from multiple types of genomic data is a considerable problem in the bioinformatics research. However, most of the existing methods did not fully use the experimentally validated miRNA-mRNA interactions. Here, we developed RMLM and RMLMSe to predict the relationship between miRNAs and their targets. RMLM and RMLMSe are global approaches as they can reconstruct the missing associations for all the miRNA-target simultaneously and RMLMSe demonstrates that the integration of sequence information can improve the performance of RMLM. In RMLM, we use RM measure to evaluate different relatedness between miRNA and its target based on different meta-paths; logistic regression and MLE method are employed to estimate the weight of different meta-paths. In RMLMSe, sequence information is utilized to improve the performance of RMLM. Here, we carry on fivefold cross validation and pathway enrichment analysis to prove the performance of our methods. The fivefold experiments show that our methods have higher AUC scores compared with other methods and the integration of sequence information can improve the performance of miRNA-target association prediction.

  13. MAGIA, a web-based tool for miRNA and Genes Integrated Analysis. (United States)

    Sales, Gabriele; Coppe, Alessandro; Bisognin, Andrea; Biasiolo, Marta; Bortoluzzi, Stefania; Romualdi, Chiara


    MAGIA (miRNA and genes integrated analysis) is a novel web tool for the integrative analysis of target predictions, miRNA and gene expression data. MAGIA is divided into two parts: the query section allows the user to retrieve and browse updated miRNA target predictions computed with a number of different algorithms (PITA, miRanda and Target Scan) and Boolean combinations thereof. The analysis section comprises a multistep procedure for (i) direct integration through different functional measures (parametric and non-parametric correlation indexes, a variational Bayesian model, mutual information and a meta-analysis approach based on P-value combination) of mRNA and miRNA expression data, (ii) construction of bipartite regulatory network of the best miRNA and mRNA putative interactions and (iii) retrieval of information available in several public databases of genes, miRNAs and diseases and via scientific literature text-mining. MAGIA is freely available for Academic users at

  14. Object-based Analysis for Extraction of Dominant Tree Species

    Institute of Scientific and Technical Information of China (English)

    Meiyun; SHAO; Xia; JING; Lu; WANG


    As forest is of great significance for our whole development and the sustainable plan is so focus on it. It is very urgent for us to have the whole distribution,stock volume and other related information about that. So the forest inventory program is on our schedule. Aiming at dealing with the problem in extraction of dominant tree species,we tested the highly hot method-object-based analysis. Based on the ALOS image data,we combined multi-resolution in e Cognition software and fuzzy classification algorithm. Through analyzing the segmentation results,we basically extract the spruce,the pine,the birch and the oak of the study area. Both the spectral and spatial characteristics were derived from those objects,and with the help of GLCM,we got the differences of each species. We use confusion matrix to do the Classification accuracy assessment compared with the actual ground data and this method showed a comparatively good precision as 87% with the kappa coefficient 0. 837.

  15. Data Clustering Analysis Based on Wavelet Feature Extraction

    Institute of Scientific and Technical Information of China (English)

    QIANYuntao; TANGYuanyan


    A novel wavelet-based data clustering method is presented in this paper, which includes wavelet feature extraction and cluster growing algorithm. Wavelet transform can provide rich and diversified information for representing the global and local inherent structures of dataset. therefore, it is a very powerful tool for clustering feature extraction. As an unsupervised classification, the target of clustering analysis is dependent on the specific clustering criteria. Several criteria that should be con-sidered for general-purpose clustering algorithm are pro-posed. And the cluster growing algorithm is also con-structed to connect clustering criteria with wavelet fea-tures. Compared with other popular clustering methods,our clustering approach provides multi-resolution cluster-ing results,needs few prior parameters, correctly deals with irregularly shaped clusters, and is insensitive to noises and outliers. As this wavelet-based clustering method isaimed at solving two-dimensional data clustering prob-lem, for high-dimensional datasets, self-organizing mapand U-matrlx method are applied to transform them intotwo-dimensional Euclidean space, so that high-dimensional data clustering analysis,Results on some sim-ulated data and standard test data are reported to illus-trate the power of our method.

  16. Extracting Communities of Interests for Semantics-Based Graph Searches (United States)

    Nakatsuji, Makoto; Tanaka, Akimichi; Uchiyama, Toshio; Fujimura, Ko

    Users recently find their interests by checking the contents published or mentioned by their immediate neighbors in social networking services. We propose semantics-based link navigation; links guide the active user to potential neighbors who may provide new interests. Our method first creates a graph that has users as nodes and shared interests as links. Then it divides the graph by link pruning to extract practical numbers, that the active user can navigate, of interest-sharing groups, i.e. communities of interests (COIs). It then attaches a different semantic tag to the link to each representative user, which best reflects the interests of COIs that they are included in, and to the link to each immediate neighbor of the active user. It finally calculates link attractiveness by analyzing the semantic tags on links. The active user can select the link to access by checking the semantic tags and link attractiveness. User interests extracted from large scale actual blog-entries are used to confirm the efficiency of our proposal. Results show that navigation based on link attractiveness and representative users allows the user to find new interests much more accurately than is otherwise possible.

  17. Liquid-phase extraction coupled with metal-organic frameworks-based dispersive solid phase extraction of herbicides in peanuts. (United States)

    Li, Na; Wang, Zhibing; Zhang, Liyuan; Nian, Li; Lei, Lei; Yang, Xiao; Zhang, Hanqi; Yu, Aimin


    Liquid-phase extraction coupled with metal-organic frameworks-based dispersive solid phase extraction was developed and applied to the extraction of pesticides in high fatty matrices. The herbicides were ultrasonically extracted from peanut using ethyl acetate as extraction solvent. The separation of the analytes from a large amount of co-extractive fat was achieved by dispersive solid-phase extraction using MIL-101(Cr) as sorbent. In this step, the analytes were adsorbed on MIL-101(Cr) and the fat remained in bulk. The herbicides were separated and determined by high-performance liquid chromatography. The experimental parameters, including type and volume of extraction solvent, ultrasonication time, volume of hexane and eluting solvent, amount of MIL-101(Cr) and dispersive solid phase extraction time, were optimized. The limits of detection for herbicides range from 0.98 to 1.9 μg/kg. The recoveries of the herbicides are in the range of 89.5-102.7% and relative standard deviations are equal or lower than 7.0%. The proposed method is simple, effective and suitable for treatment of the samples containing high content of fat.

  18. miRFam: an effective automatic miRNA classification method based on n-grams and a multiclass SVM

    Directory of Open Access Journals (Sweden)

    Zhou Shuigeng


    Full Text Available Abstract Background MicroRNAs (miRNAs are ~22 nt long integral elements responsible for post-transcriptional control of gene expressions. After the identification of thousands of miRNAs, the challenge is now to explore their specific biological functions. To this end, it will be greatly helpful to construct a reasonable organization of these miRNAs according to their homologous relationships. Given an established miRNA family system (e.g. the miRBase family organization, this paper addresses the problem of automatically and accurately classifying newly found miRNAs to their corresponding families by supervised learning techniques. Concretely, we propose an effective method, miRFam, which uses only primary information of pre-miRNAs or mature miRNAs and a multiclass SVM, to automatically classify miRNA genes. Results An existing miRNA family system prepared by miRBase was downloaded online. We first employed n-grams to extract features from known precursor sequences, and then trained a multiclass SVM classifier to classify new miRNAs (i.e. their families are unknown. Comparing with miRBase's sequence alignment and manual modification, our study shows that the application of machine learning techniques to miRNA family classification is a general and more effective approach. When the testing dataset contains more than 300 families (each of which holds no less than 5 members, the classification accuracy is around 98%. Even with the entire miRBase15 (1056 families and more than 650 of them hold less than 5 samples, the accuracy surprisingly reaches 90%. Conclusions Based on experimental results, we argue that miRFam is suitable for application as an automated method of family classification, and it is an important supplementary tool to the existing alignment-based small non-coding RNA (sncRNA classification methods, since it only requires primary sequence information. Availability The source code of miRFam, written in C++, is freely and publicly

  19. Spatially Enhanced Differential RNA Methylation Analysis from Affinity-Based Sequencing Data with Hidden Markov Model. (United States)

    Zhang, Yu-Chen; Zhang, Shao-Wu; Liu, Lian; Liu, Hui; Zhang, Lin; Cui, Xiaodong; Huang, Yufei; Meng, Jia


    With the development of new sequencing technology, the entire N6-methyl-adenosine (m(6)A) RNA methylome can now be unbiased profiled with methylated RNA immune-precipitation sequencing technique (MeRIP-Seq), making it possible to detect differential methylation states of RNA between two conditions, for example, between normal and cancerous tissue. However, as an affinity-based method, MeRIP-Seq has yet provided base-pair resolution; that is, a single methylation site determined from MeRIP-Seq data can in practice contain multiple RNA methylation residuals, some of which can be regulated by different enzymes and thus differentially methylated between two conditions. Since existing peak-based methods could not effectively differentiate multiple methylation residuals located within a single methylation site, we propose a hidden Markov model (HMM) based approach to address this issue. Specifically, the detected RNA methylation site is further divided into multiple adjacent small bins and then scanned with higher resolution using a hidden Markov model to model the dependency between spatially adjacent bins for improved accuracy. We tested the proposed algorithm on both simulated data and real data. Result suggests that the proposed algorithm clearly outperforms existing peak-based approach on simulated systems and detects differential methylation regions with higher statistical significance on real dataset.

  20. 三种方法提取血清miRNA效果的比较%Comparing the serum miRNA extraction effect of three methods

    Institute of Scientific and Technical Information of China (English)

    陈昌国; 陈秋圆; 郭建巍; 马志家; 刘敏; 赵强元; 张雅芳


    目的:比较Trizol法、蛋白酶K消化+ Trizol法及沉淀裂解法提取血清miRNA的效果。方法分别选取2例健康人、2例肺炎患者和2例肺癌患者血清,以Let‐7a‐5p作为目标mRNA ,线虫Cel‐miR‐39‐3p作为内参,分别采用Trizol法、蛋白酶K消化+ Trizol法及沉淀裂解法提取血清miRNA ,以Let‐7a‐5p及Cel‐miR‐39‐3p特异性引物进行反转录PCR ,以反转录PCR产物为模板运用SYBGreen Ⅰ法进行荧光定量PCR检测。结果 T rizol法与蛋白酶K消化+ Trizol法均能有效提取到血清miRNA ,而沉淀裂解法由于没有对血浆中的RNA进行富集故提取效果不佳。T rizol法提取较蛋白酶K消化+ T rizol法相比,可以减少蛋白酶K的消化时间且提取效果更好,并且血清与Trizol试剂的体积比为200∶600时效果相对较好。三种方法检测Cel‐miR‐39‐3p的Ct值基本一致。Tr‐izol法和蛋白酶K消化+ Trizol法在提取过程中加入的内参丢失较少。结论 Trizol法作为总RNA提取的手段适用于血清miRNA提取,通过调整血清与Trizol试剂的用量可使提取到的血清miRNA能够满足试验需要。%Objective To compare the serum miRNA extracting efficiency of Trizol method ,protease K diges‐tion combined with Trizol method and precipitation pyrolysis method .Methods 2 healthy persons ,2 pneumonia pa‐tients and 2 lung cancer patients were enrolled in the study and their serum samples were collected .miRNA were ex‐tracted from serum samples respectively by using Trizol method ,protease K digestion combined with Trizol method and precipitation pyrolysis method .Let‐7a‐5p was the objective miRNA of detection ,with Cel‐miR‐39‐3p as the incor‐poration of spike‐in reference .The specific primers of Let‐7a‐5p (objective miRNA) and Cel‐miR‐39‐3p (internal ref‐erence) were used in reverse transcription PCR .The products of reverse transcription PCR were used as

  1. Epithelial-mesenchymal transition and cancer stem cells, mediated by a long non-coding RNA, HOTAIR, are involved in cell malignant transformation induced by cigarette smoke extract

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Yi; Luo, Fei; Xu, Yuan; Wang, Bairu; Zhao, Yue; Xu, Wenchao; Shi, Le; Lu, Xiaolin; Liu, Qizhan, E-mail:


    The incidence of lung diseases, including cancer, caused by cigarette smoke is increasing, but the molecular mechanisms of gene regulation induced by cigarette smoke remain unclear. This report describes a long noncoding RNA (lncRNA) that is induced by cigarette smoke extract (CSE) and experiments utilizing lncRNAs to integrate inflammation with the epithelial-mesenchymal transition (EMT) in human bronchial epithelial (HBE) cells. The present study shows that, induced by CSE, IL-6, a pro-inflammatory cytokine, leads to activation of STAT3, a transcription activator. A ChIP assay determined that the interaction of STAT3 with the promoter regions of HOX transcript antisense RNA (HOTAIR) increased levels of HOTAIR. Blocking of IL-6 with anti-IL-6 antibody, decreasing STAT3, and inhibiting STAT3 activation reduced HOTAIR expression. Moreover, for HBE cells cultured in the presence of HOTAIR siRNA for 24 h, the CSE-induced EMT, formation of cancer stem cells (CSCs), and malignant transformation were reversed. Thus, IL-6, acting on STAT3 signaling, which up-regulates HOTAIR in an autocrine manner, contributes to the EMT and to CSCs induced by CSE. These data define a link between inflammation and EMT, processes involved in the malignant transformation of cells caused by CSE. This link, mediated through lncRNAs, establishes a mechanism for CSE-induced lung carcinogenesis. - Highlights: • STAT3 directly regulates the levels of LncRNA HOTAIR. • LncRNA HOTAIR mediates the link between inflammation and EMT. • LncRNA HOTAIR is involved in the malignant transformation of cells caused by CSE.

  2. Knowledge-based reasoning to annotate noncoding RNA using multi-agent system. (United States)

    Arruda, Wosley C; Souza, Daniel S; Ralha, Célia G; Walter, Maria Emilia M T; Raiol, Tainá; Brigido, Marcelo M; Stadler, Peter F


    Noncoding RNAs (ncRNAs) have been focus of intense research over the last few years. Since characteristics and signals of ncRNAs are not entirely known, researchers use different computational tools together with their biological knowledge to predict putative ncRNAs. In this context, this work presents ncRNA-Agents, a multi-agent system to annotate ncRNAs based on the output of different tools, using inference rules to simulate biologists' reasoning. Experiments with data from the fungus Saccharomyces cerevisiae allowed to measure the performance of ncRNA-Agents, with better sensibility, when compared to Infernal, a widely used tool for annotating ncRNA. Besides, data of the Schizosaccharomyces pombe and Paracoccidioides brasiliensis fungi identified novel putative ncRNAs, which demonstrated the usefulness of our approach. NcRNA-Agents can be be found at:

  3. Targeting MicroRNA in Cancer Using Plant-Based Proanthocyanidins

    Directory of Open Access Journals (Sweden)

    Rishipal R. Bansode


    Full Text Available Proanthocyanidins are oligomeric flavonoids found in plant sources, most notably in apples, cinnamon, grape skin and cocoa beans. They have been also found in substantial amounts in cranberry, black currant, green tea, black tea and peanut skins. These compounds have been recently investigated for their health benefits. Proanthocyanidins have been demonstrated to have positive effects on various metabolic disorders such as inflammation, obesity, diabetes and insulin resistance. Another upcoming area of research that has gained widespread interest is microRNA (miRNA-based anticancer therapies. MicroRNAs are short non-coding RNA segments, which plays a crucial role in RNA silencing and post-transcriptional regulation of gene expression. Currently, miRNA based anticancer therapies are being investigated either alone or in combination with current treatment methods. In this review, we summarize the current knowledge and investigate the potential of naturally occurring proanthocyanidins in modulating miRNA expression. We will also assess the strategies and challenges of using this approach as potential cancer therapeutics.

  4. Probing of tertiary interactions in RNA: 2'-hydroxyl-base contacts between the RNase P RNA and pre-tRNA. (United States)

    Pan, T; Loria, A; Zhong, K


    A general method has been developed to analyze all 2' hydroxyl groups involved in tertiary interactions in RNA in a single experiment. This method involves comparing the activity of populations of circularly permuted RNAs that contain or lack potential hydrogen-bond donors at each position. The 2' hydroxyls of the pre-tRNA substrate identified as potential hydrogen bond donors in intermolecular interactions with the ribozyme from eubacterial RNase P (P RNA) are located in the T stem and T loop, acceptor stem, and 3' CCA regions. To locate the hydrogen-bond acceptors for one of those 2' hydroxyls in the P RNA, a phylogenetically conserved adenosine was mutated to a guanosine. When this mutant P RNA was used, increased cleavage activity of a single circularly permuted substrate within the population was observed. The cleavage efficiency (kcat/Km) of a singly 2'-deoxy-substituted substrate at this position in the T stem was also determined. For the wild-type P RNA, the catalytic efficiency was significantly decreased compared with that of the all-ribo substrate, consistent with the notion that this 2' hydroxyl plays an important role. For the P RNA mutant, no additional effect was found upon 2'-deoxy substitution. We propose that this particular 2' hydroxyl in the pre-tRNA interacts specifically with this adenosine in the P RNA. This method should be useful in examining the role of 2' hydroxyl groups in other RNA-RNA and RNA-protein complexes. Images Fig. 2 Fig. 3 Fig. 4 PMID:8618931

  5. Identification of co-expression gene networks, regulatory genes and pathways for obesity based on adipose tissue RNA Sequencing in a porcine model

    DEFF Research Database (Denmark)

    Kogelman, Lisette; Cirera Salicio, Susanna; Zhernakova, Daria V.


    interactions. Identification of co-expressed and regulatory genes in RNA extracted from relevant tissues representing lean and obese individuals provides an entry point for the identification of genes and pathways of importance to the development of obesity. The pig, an omnivorous animal, is an excellent model...... in a porcine model. Methods We selected 36 animals for RNA Sequencing from a previously created F2 pig population representing three extreme groups based on their predicted genetic risks for obesity. We applied Weighted Gene Co-expression Network Analysis (WGCNA) to detect clusters of highly co-expressed genes...... in humans and rodents, e.g. CSF1R and MARC2. Conclusions To our knowledge, this is the first study to apply systems biology approaches using porcine adipose tissue RNA-Sequencing data in a genetically characterized porcine model for obesity. We revealed complex networks, pathways, candidate and regulatory...

  6. Probing electronic coupling between adenine bases in RNA strands from synchrotron radiation circular dichroism experiments

    DEFF Research Database (Denmark)

    Nielsen, Lisbeth Munksgård; Hoffmann, Søren Vrønning; Nielsen, Steen Brøndsted


    Circular dichroism spectra (176–330 nm) of RNA adenine oligomers, (rA)n (n = 1–10, 12, 15, and 20), reveal electronic coupling between two bases in short strands. The number of interacting bases in long strands is more and larger than that reported previously for the corresponding DNA strands....

  7. 少白细胞血小板miRNA提取方法*%Method of extraction of miRNA from human leucocyte depleted-platelet

    Institute of Scientific and Technical Information of China (English)

    罗茂; 万沁; 刘飞; 马卫中; 吴剑波


    Objective: To establish a simple and convenient method isolation of high-quality miRNA from human leukocyte-depleted platelet preparation. Methods: Small RNA in healthy platelets was extracted by conv-entional gradient centrifugation, MACS and the mirVanaTM miRNA Isolation Kit. Leukocyte contamination in the final purified platelet preparation was estimated by conventional gradient centrifugation, platelet counting and the transcript levels of the gene encoding CD45/GPIIb marker in platelet preparations. The concentration, purity and integrity were detected, and a miRNA' cDNA library which was constructed by RNA-tailing and primer-extension reverse transcription (RT)-PCR was used as the template for the amplification of U6 and miR-449b to verify the authenticity. Results:Small RNA was extracted with high purity, high efficiency of leukocyte depletion, Its cDNA had high authenticity despite its low concentration. Conclusion:A method of extraction of microRNA from human platelet preparation with high efficiency of leukocyte depletion, and high quality of miRNA' cDNA library is constructed, which is applicative to future miRNA experiments.%  目的:建立常规梯度离心结合MACS磁珠分选纯化和小分子RNA分离试剂盒简便快速提取血小板miRNA的方法。方法:利用常规梯度离心获取血小板后,结合MACS磁珠分选和小分子RNA分离试剂盒提取少白细胞血小板(LDP)小分子RNA;经血小板计数、CD45和GPIIb mRNA marker PCR扩增检测,判定纯化效果;通过小分子RNA浓度、纯度及毛细管电泳完整性检测,以及内参U6和血小板miR-449b加尾及引物延伸RT-PCR后PCR扩增结果验证构建血小板miRNA的cDNA模板真实性。结果:提取小分子RNA纯度较高,有效去除白细胞污染,虽然含量较少,但内参U6和血小板miR-449b真实性验证结果提示,构建血小板miRNA的cDNA模板质量较高。结论:常规梯度离心结合MACS磁珠分选纯化和小分子RNA

  8. An atlas of RNA base pairs involving modified nucleobases with optimal geometries and accurate energies

    KAUST Repository

    Chawla, Mohit


    Posttranscriptional modifications greatly enhance the chemical information of RNA molecules, contributing to explain the diversity of their structures and functions. A significant fraction of RNA experimental structures available to date present modified nucleobases, with half of them being involved in H-bonding interactions with other bases, i.e. ‘modified base pairs’. Herein we present a systematic investigation of modified base pairs, in the context of experimental RNA structures. To this end, we first compiled an atlas of experimentally observed modified base pairs, for which we recorded occurrences and structural context. Then, for each base pair, we selected a representative for subsequent quantum mechanics calculations, to find out its optimal geometry and interaction energy. Our structural analyses show that most of the modified base pairs are non Watson–Crick like and are involved in RNA tertiary structure motifs. In addition, quantum mechanics calculations quantify and provide a rationale for the impact of the different modifications on the geometry and stability of the base pairs they participate in.

  9. Controlled RNA contamination and degradation and its impact on qPCR gene expression in S. epidermidis biofilms



    RNA quality is of utmost importance to perform gene expression quantification by qPCR. The classical methods used to determine \\RNA\\ quality are based on electrophoresis and spectrophotometer assessment, namely A260/A280 and A260/A230 ratios. It was previously shown that due to the complex nature of Staphylococcus epidermidis biofilms, \\RNA\\ extraction procedures could impact mRNA quality and thus accurate quantification. Herein, we contaminated and degraded \\RNA\\ extracted from S. epidermidi...

  10. Diagnosis-Therapy Integrative Systems Based on Magnetic RNA Nanoflowers for Co-drug Delivery and Targeted Therapy. (United States)

    Guo, Yingshu; Li, Shuang; Wang, Yujie; Zhang, Shusheng


    This study was to develop a codrug delivery system for targeting cancer therapy based on magnetic RNA nanoflowers (RNA NF). Compared with traditional nucleic acid structure, convenient separation can be achieved by introducing magnetic nanoparticle (MNP) into RNA NF. Folic acid (FA) modified MNP/RNA NF (FA/MNP/RNA NF) was used as a targeting nanocarrier with excellent biocompatibility to overcome the nonselectivity of MNP/RNA NF. And then, anticancer drug doxorubicin (DOX) and photosensitizer 5, 10, 15, 20-tetrakis (1-methylpyridinium-4-yl) porphyrin (TMPyP4) binding with RNA NF were used as codrug cargo models. RNA NF was first used for codrug delivery. So, imaging fluorescent tags, target recognition element, and drug molecules were all assembled together on the surface of MNP/RNA NF. The experimental results suggested that the treatment efficacy of codrug delivery platform (FA/MNP/RNA NF/D/T) was better than single-drug delivery platform (FA/MNP/RNA NF/D). Besides, the FA/MNP/RNA NF was used as a probe for cancer cell detection. The limit of detection was 50 HeLa cells. In conclusion, the codrug delivery platform based on FA/MNP/RNA NF was a promising approach for the intracellular quantification of other biomolecules, as well as a diagnosis-therapy integrative system.

  11. Vision-Based Faint Vibration Extraction Using Singular Value Decomposition

    Directory of Open Access Journals (Sweden)

    Xiujun Lei


    Full Text Available Vibration measurement is important for understanding the behavior of engineering structures. Unlike conventional contact-type measurements, vision-based methodologies have attracted a great deal of attention because of the advantages of remote measurement, nonintrusive characteristic, and no mass introduction. It is a new type of displacement sensor which is convenient and reliable. This study introduces the singular value decomposition (SVD methods for video image processing and presents a vibration-extracted algorithm. The algorithms can successfully realize noncontact displacement measurements without undesirable influence to the structure behavior. SVD-based algorithm decomposes a matrix combined with the former frames to obtain a set of orthonormal image bases while the projections of all video frames on the basis describe the vibration information. By means of simulation, the parameters selection of SVD-based algorithm is discussed in detail. To validate the algorithm performance in practice, sinusoidal motion tests are performed. Results indicate that the proposed technique can provide fairly accurate displacement measurement. Moreover, a sound barrier experiment showing how the high-speed rail trains affect the sound barrier nearby is carried out. It is for the first time to be realized at home and abroad due to the challenge of measuring environment.

  12. COSMO-RS-based extractant screening for phenol extraction as model system

    NARCIS (Netherlands)

    Burghoff, B.; Goetheer, E.L.V.; Haan, A.B. de


    The focus of this investigation is the development of a fast and reliable extractant screening approach. Phenol extraction is selected as the model process. A quantum chemical conductor-like screening model for real solvents (COSMO-RS) is combined with molecular design considerations. For this purpo

  13. Optimization of RNA-based c-di-GMP fluorescent sensors through tuning their structural modules. (United States)

    Inuzuka, Saki; Matsumura, Shigeyoshi; Ikawa, Yoshiya


    Cyclic diguanylate (c-di-GMP) is a second messenger of bacteria and its detection is an important issue in basic and applied microbiology. As c-di-GMP riboswitch ligand-binding domains (aptamer domains) capture c-di-GMP with high affinity and selectivity, they are promising platforms for the development of RNA-based c-di-GMP sensors. We analyzed two previously reported c-di-GMP sensor RNAs derived from the Vc2 riboswitch. We also designed and tested their variants, some of which showed improved properties as RNA-based c-di-GMP sensors.

  14. URS DataBase: universe of RNA structures and their motifs. (United States)

    Baulin, Eugene; Yacovlev, Victor; Khachko, Denis; Spirin, Sergei; Roytberg, Mikhail


    The Universe of RNA Structures DataBase (URSDB) stores information obtained from all RNA-containing PDB entries (2935 entries in October 2015). The content of the database is updated regularly. The database consists of 51 tables containing indexed data on various elements of the RNA structures. The database provides a web interface allowing user to select a subset of structures with desired features and to obtain various statistical data for a selected subset of structures or for all structures. In particular, one can easily obtain statistics on geometric parameters of base pairs, on structural motifs (stems, loops, etc.) or on different types of pseudoknots. The user can also view and get information on an individual structure or its selected parts, e.g. RNA-protein hydrogen bonds. URSDB employs a new original definition of loops in RNA structures. That definition fits both pseudoknot-free and pseudoknotted secondary structures and coincides with the classical definition in case of pseudoknot-free structures. To our knowledge, URSDB is the first database supporting searches based on topological classification of pseudoknots and on extended loop classification.Database URL:

  15. Mechanism of gold solvent extraction from aurocyanide solution by quaternary amines: models of extracting species based on hydrogen bonding

    Institute of Scientific and Technical Information of China (English)

    马刚; 闫文飞; 陈景; 严纯华; 高宏成; 周维金; 施鼐; 吴谨光; 徐光宪; 黄昆; 余建民; 崔宁


    The mechanism of gold solvent extraction from KAu(CN)2 solution was investigated by means of FTIR, EXAFS, ICP and radioactive tracer methods. Two extraction systems were studied, namely N263-tributyl phosphate(TBP)-n-dodecane and N263-iso-octanol-n-dodecane. High-resolution FT IR spectroscopy indicated that the CN stretching vibrations of the two extraction systems differred greatly. In order to interpret the significant difference in CN stretching vibrations, twoextracting species models are proposed——supramolecular structures based on the formation ofhydrogen bonds between Au(CN)2- and modifiers such as TBP and iso-octanol.

  16. An image segmentation based method for iris feature extraction

    Institute of Scientific and Technical Information of China (English)

    XU Guang-zhu; ZHANG Zai-feng; MA Yi-de


    In this article, the local anomalistic blocks such ascrypts, furrows, and so on in the iris are initially used directly asiris features. A novel image segmentation method based onintersecting cortical model (ICM) neural network was introducedto segment these anomalistic blocks. First, the normalized irisimage was put into ICM neural network after enhancement.Second, the iris features were segmented out perfectly and wereoutput in binary image type by the ICM neural network. Finally,the fourth output pulse image produced by ICM neural networkwas chosen as the iris code for the convenience of real timeprocessing. To estimate the performance of the presentedmethod, an iris recognition platform was produced and theHamming Distance between two iris codes was computed tomeasure the dissimilarity between them. The experimentalresults in CASIA vl.0 and Bath iris image databases show thatthe proposed iris feature extraction algorithm has promisingpotential in iris recognition.

  17. Transmission line icing prediction based on DWT feature extraction (United States)

    Ma, T. N.; Niu, D. X.; Huang, Y. L.


    Transmission line icing prediction is the premise of ensuring the safe operation of the network as well as the very important basis for the prevention of freezing disasters. In order to improve the prediction accuracy of icing, a transmission line icing prediction model based on discrete wavelet transform (DWT) feature extraction was built. In this method, a group of high and low frequency signals were obtained by DWT decomposition, and were fitted and predicted by using partial least squares regression model (PLS) and wavelet least square support vector model (w-LSSVM). Finally, the final result of the icing prediction was obtained by adding the predicted values of the high and low frequency signals. The results showed that the method is effective and feasible in the prediction of transmission line icing.

  18. An answer summarization method based on keyword extraction

    Directory of Open Access Journals (Sweden)

    Fan Qiaoqing


    Full Text Available In order to reduce the redundancy of answer summary generated from community q&a dataset without topic tags, we propose an answer summarization algorithm based on keyword extraction. We combine tf-idf with word vector to change the influence transferred ratio equation in TextRank. And then during summarizing, we take the ratio of the number of sentences containing any keyword to the total number of candidate sentences as an adaptive factor for AMMR. Meanwhile we reuse the scores of keywords generated by TextRank as a weight factor for sentence similarity computing. Experimental results show that the proposed answer summarization is better than the traditional MMR and AMMR.

  19. Layering-based Breakpoint Handling in Contour Line Extraction

    Institute of Scientific and Technical Information of China (English)

    CHEN Dan; LONG Yi; CAI Jinhua


    This paper deals withthe automatic connection of contourlines extracted from a scanned browngeographical map. For the variety oftopographical elements contained on amap, the factors causing the interrup-tion of contour line are also multiform,which make the connection task verydifficult. On the basis of separatingthose elements always making the con-tours break and regarding them as ref-erent layers, a layering-based methodis presented. The purpose is to takeinto account property information (likeinclination and configuration) of con-tour lines when they come across otherdifferent symbols, such as gully, cliff,dry land and elevation annotation etc.In this paper, the authors propose thatit should be far more effective and di-rect to adopt different algorithmic op-erators to different factors than usingsingle one operator to all.

  20. The selective tRNA aminoacylation mechanism based on a single G•U pair. (United States)

    Naganuma, Masahiro; Sekine, Shun-ichi; Chong, Yeeting Esther; Guo, Min; Yang, Xiang-Lei; Gamper, Howard; Hou, Ya-Ming; Schimmel, Paul; Yokoyama, Shigeyuki


    Ligation of tRNAs with their cognate amino acids, by aminoacyl-tRNA synthetases, establishes the genetic code. Throughout evolution, tRNA(Ala) selection by alanyl-tRNA synthetase (AlaRS) has depended predominantly on a single wobble base pair in the acceptor stem, G3•U70, mainly on the kcat level. Here we report the crystal structures of an archaeal AlaRS in complex with tRNA(Ala) with G3•U70 and its A3•U70 variant. AlaRS interacts with both the minor- and the major-groove sides of G3•U70, widening the major groove. The geometry difference between G3•U70 and A3•U70 is transmitted along the acceptor stem to the 3'-CCA region. Thus, the 3'-CCA region of tRNA(Ala) with G3•U70 is oriented to the reactive route that reaches the active site, whereas that of the A3•U70 variant is folded back into the non-reactive route. This novel mechanism enables the single wobble pair to dominantly determine the specificity of tRNA selection, by an approximate 100-fold difference in kcat.

  1. Logic integration of mRNA signals by an RNAi-based molecular computer. (United States)

    Xie, Zhen; Liu, Siyuan John; Bleris, Leonidas; Benenson, Yaakov


    Synthetic in vivo molecular 'computers' could rewire biological processes by establishing programmable, non-native pathways between molecular signals and biological responses. Multiple molecular computer prototypes have been shown to work in simple buffered solutions. Many of those prototypes were made of DNA strands and performed computations using cycles of annealing-digestion or strand displacement. We have previously introduced RNA interference (RNAi)-based computing as a way of implementing complex molecular logic in vivo. Because it also relies on nucleic acids for its operation, RNAi computing could benefit from the tools developed for DNA systems. However, these tools must be harnessed to produce bioactive components and be adapted for harsh operating environments that reflect in vivo conditions. In a step toward this goal, we report the construction and implementation of biosensors that 'transduce' mRNA levels into bioactive, small interfering RNA molecules via RNA strand exchange in a cell-free Drosophila embryo lysate, a step beyond simple buffered environments. We further integrate the sensors with our RNAi 'computational' module to evaluate two-input logic functions on mRNA concentrations. Our results show how RNA strand exchange can expand the utility of RNAi computing and point toward the possibility of using strand exchange in a native biological setting.

  2. Predicting the functions of long noncoding RNAs using RNA-seq based on Bayesian network. (United States)

    Xiao, Yun; Lv, Yanling; Zhao, Hongying; Gong, Yonghui; Hu, Jing; Li, Feng; Xu, Jinyuan; Bai, Jing; Yu, Fulong; Li, Xia


    Long noncoding RNAs (lncRNAs) have been shown to play key roles in various biological processes. However, functions of most lncRNAs are poorly characterized. Here, we represent a framework to predict functions of lncRNAs through construction of a regulatory network between lncRNAs and protein-coding genes. Using RNA-seq data, the transcript profiles of lncRNAs and protein-coding genes are constructed. Using the Bayesian network method, a regulatory network, which implies dependency relations between lncRNAs and protein-coding genes, was built. In combining protein interaction network, highly connected coding genes linked by a given lncRNA were subsequently used to predict functions of the lncRNA through functional enrichment. Application of our method to prostate RNA-seq data showed that 762 lncRNAs in the constructed regulatory network were assigned functions. We found that lncRNAs are involved in diverse biological processes, such as tissue development or embryo development (e.g., nervous system development and mesoderm development). By comparison with functions inferred using the neighboring gene-based method and functions determined using lncRNA knockdown experiments, our method can provide comparable predicted functions of lncRNAs. Overall, our method can be applied to emerging RNA-seq data, which will help researchers identify complex relations between lncRNAs and coding genes and reveal important functions of lncRNAs.

  3. tRNA acceptor-stem and anticodon bases embed separate features of amino acid chemistry. (United States)

    Carter, Charles W; Wolfenden, Richard


    The universal genetic code is a translation table by which nucleic acid sequences can be interpreted as polypeptides with a wide range of biological functions. That information is used by aminoacyl-tRNA synthetases to translate the code. Moreover, amino acid properties dictate protein folding. We recently reported that digital correlation techniques could identify patterns in tRNA identity elements that govern recognition by synthetases. Our analysis, and the functionality of truncated synthetases that cannot recognize the tRNA anticodon, support the conclusion that the tRNA acceptor stem houses an independent code for the same 20 amino acids that likely functioned earlier in the emergence of genetics. The acceptor-stem code, related to amino acid size, is distinct from a code in the anticodon that is related to amino acid polarity. Details of the acceptor-stem code suggest that it was useful in preserving key properties of stereochemically-encoded peptides that had developed the capacity to interact catalytically with RNA. The quantitative embedding of the chemical properties of amino acids into tRNA bases has implications for the origins of molecular biology.

  4. DIANA-TarBase v7.0: indexing more than half a million experimentally supported miRNA:mRNA interactions. (United States)

    Vlachos, Ioannis S; Paraskevopoulou, Maria D; Karagkouni, Dimitra; Georgakilas, Georgios; Vergoulis, Thanasis; Kanellos, Ilias; Anastasopoulos, Ioannis-Laertis; Maniou, Sofia; Karathanou, Konstantina; Kalfakakou, Despina; Fevgas, Athanasios; Dalamagas, Theodore; Hatzigeorgiou, Artemis G


    microRNAs (miRNAs) are short non-coding RNA species, which act as potent gene expression regulators. Accurate identification of miRNA targets is crucial to understanding their function. Currently, hundreds of thousands of miRNA:gene interactions have been experimentally identified. However, this wealth of information is fragmented and hidden in thousands of manuscripts and raw next-generation sequencing data sets. DIANA-TarBase was initially released in 2006 and it was the first database aiming to catalog published experimentally validated miRNA:gene interactions. DIANA-TarBase v7.0 ( aims to provide for the first time hundreds of thousands of high-quality manually curated experimentally validated miRNA:gene interactions, enhanced with detailed meta-data. DIANA-TarBase v7.0 enables users to easily identify positive or negative experimental results, the utilized experimental methodology, experimental conditions including cell/tissue type and treatment. The new interface provides also advanced information ranging from the binding site location, as identified experimentally as well as in silico, to the primer sequences used for cloning experiments. More than half a million miRNA:gene interactions have been curated from published experiments on 356 different cell types from 24 species, corresponding to 9- to 250-fold more entries than any other relevant database. DIANA-TarBase v7.0 is freely available.

  5. Chaperone probes and bead-based enhancement to improve the direct detection of mRNA using silicon photonic sensor arrays. (United States)

    Kindt, Jared T; Bailey, Ryan C


    Herein, we describe the utility of chaperone probes and a bead-based signal enhancement strategy for the analysis of full length mRNA transcripts using arrays of silicon photonic microring resonators. Changes in the local refractive index near microring sensors associated with biomolecular binding events are transduced as a shift in the resonant wavelength supported by the cavity, enabling the sensitive analysis of numerous analytes of interest. We employ the sensing platform for both the direct and bead-enhanced detection of three different mRNA transcripts, achieving a dynamic range spanning over 4 orders of magnitude and demonstrating expression profiling capabilities in total RNA extracts from the HL-60 cell line. Small, dual-use DNA chaperone molecules were developed and found to both enhance the binding kinetics of mRNA transcripts by disrupting complex secondary structure and serve as sequence-specific linkers for subsequent bead amplification. Importantly, this approach does not require amplification of the mRNA transcript, thereby allowing for simplified analyses that do not require expensive enzymatic reagents or temperature ramping capabilities associated with RT-PCR-based methods.

  6. Plant microRNA-target interaction identification model based on the integration of prediction tools and support vector machine.

    Directory of Open Access Journals (Sweden)

    Jun Meng

    Full Text Available Confident identification of microRNA-target interactions is significant for studying the function of microRNA (miRNA. Although some computational miRNA target prediction methods have been proposed for plants, results of various methods tend to be inconsistent and usually lead to more false positive. To address these issues, we developed an integrated model for identifying plant miRNA-target interactions.Three online miRNA target prediction toolkits and machine learning algorithms were integrated to identify and analyze Arabidopsis thaliana miRNA-target interactions. Principle component analysis (PCA feature extraction and self-training technology were introduced to improve the performance. Results showed that the proposed model outperformed the previously existing methods. The results were validated by using degradome sequencing supported Arabidopsis thaliana miRNA-target interactions. The proposed model constructed on Arabidopsis thaliana was run over Oryza sativa and Vitis vinifera to demonstrate that our model is effective for other plant species.The integrated model of online predictors and local PCA-SVM classifier gained credible and high quality miRNA-target interactions. The supervised learning algorithm of PCA-SVM classifier was employed in plant miRNA target identification for the first time. Its performance can be substantially improved if more experimentally proved training samples are provided.

  7. Monitoring of the antiviral potential of bee venom and wax extracts against Adeno-7 (DNA) and Rift Valley fever virus (RNA) viruses models. (United States)

    Hassan, Mostafa I; Mohamed, Aly F; Amer, Moner A; Hammad, Kotb M; Riad, Saber A


    This study monitored the antiviral potential of bee venom and four wax extracts, ethanol white and black beeswax (EWW/EBW) and acetone white and black beeswax (AWW/ABW) extracts. Two different virus models namely Adeno-7 as DNA model and RVFV as RNA virus models. End point calculation assay was used to calculate virus depletion titer. The depletion of viral infectivity titer of ABW to Adeno-7 virus showed strong antiviral activity recorded a depletion of viral infectivity titer (1.66 log (10)/ ml) that gave equal action with bee venom and more than interferon IFN (1 log (10)/ ml). On the other hand, antiviral activity of EBW showed a moderate potential, while AWW showed no antiviral activity. Finally EWW showed synergetic activity against Adeno-7 virus activity. Thus, activity of wax extracts to RVFV was arranged in order of IFN bee venom > AWW & EBW > EWW and ABW recorded 3.34, 0.65, 0.5, 0.34 respectively. It is the first time to study the beeswax effect against DNA and RNA virus' models; acetone black beeswax recorded a depletion titer 1.66 log (10)/ml.

  8. RNA-Based TWIST1 Inhibition via Dendrimer Complex to Reduce Breast Cancer Cell Metastasis

    Directory of Open Access Journals (Sweden)

    James Finlay


    Full Text Available Breast cancer is the leading cause of cancer-related deaths among women in the United States, and survival rates are lower for patients with metastases and/or triple-negative breast cancer (TNBC; ER, PR, and Her2 negative. Understanding the mechanisms of cancer metastasis is therefore crucial to identify new therapeutic targets and develop novel treatments to improve patient outcomes. A potential target is the TWIST1 transcription factor, which is often overexpressed in aggressive breast cancers and is a master regulator of cellular migration through epithelial-mesenchymal transition (EMT. Here, we demonstrate an siRNA-based TWIST1 silencing approach with delivery using a modified poly(amidoamine (PAMAM dendrimer. Our results demonstrate that SUM1315 TNBC cells efficiently take up PAMAM-siRNA complexes, leading to significant knockdown of TWIST1 and EMT-related target genes. Knockdown lasts up to one week after transfection and leads to a reduction in migration and invasion, as determined by wound healing and transwell assays. Furthermore, we demonstrate that PAMAM dendrimers can deliver siRNA to xenograft orthotopic tumors and siRNA remains in the tumor for at least four hours after treatment. These results suggest that further development of dendrimer-based delivery of siRNA for TWIST1 silencing may lead to a valuable adjunctive therapy for patients with TNBC.

  9. Enzymatic synthesis and RNA interference of nucleosides incorporating stable isotopes into a base moiety. (United States)

    Hatano, Akihiko; Shiraishi, Mitsuya; Terado, Nanae; Tanabe, Atsuhiro; Fukuda, Kenji


    Thymidine phosphorylase was used to catalyze the conversion of thymidine (or methyluridine) and uracil incorporating stable isotopes to deoxyuridine (or uridine) with the uracil base incorporating the stable isotope. These base-exchange reactions proceeded with high conversion rates (75-96%), and the isolated yields were also good (64-87%). The masses of all synthetic compounds incorporating stable isotopes were identical to the theoretical molecular weights via EIMS. (13)C NMR spectra showed spin-spin coupling between (13)C and (15)N in the synthetic compounds, and the signals were split, further proving incorporation of the isotopes into the compounds. The RNA interference effects of this siRNA with uridine incorporating stable isotopes were also investigated. A 25mer siRNA had a strong knockdown effect on the MARCKS protein. The insertion position and number of uridine moieties incorporating stable isotopes introduced into the siRNA had no influence on the silencing of the target protein. This incorporation of stable isotopes into RNA and DNA has the potential to function as a chemically benign tracer in cells.

  10. A facile inhibitor screening of SARS coronavirus N protein using nanoparticle-based RNA oligonucleotide

    Directory of Open Access Journals (Sweden)

    Roh C


    Full Text Available Changhyun RohDivision of Biotechnology, Advanced Radiation Technology Institute (ARTI, Korea Atomic Energy Research Institute (KAERI, Jeongeup, Republic of KoreaAbstract: Hundreds of million people worldwide have been infected with severe acute respiratory syndrome (SARS, and the rate of global death from SARS has remarkably increased. Hence, the development of efficient drug treatments for the biological effects of SARS is highly needed. We have previously shown that quantum dots (QDs-conjugated RNA oligonucleotide is sensitive to the specific recognition of the SARS-associated coronavirus (SARS-CoV nucleocapsid (N protein. In this study, we found that a designed biochip could analyze inhibitors of the SARS-CoV N protein using nanoparticle-based RNA oligonucleotide. Among the polyphenolic compounds examined, (--catechin gallate and (--gallocatechin gallate demonstrated a remarkable inhibition activity on SARS-CoV N protein. (--catechin gallate and (--gallocatechin gallate attenuated the binding affinity in a concentrated manner as evidenced by QDs-conjugated RNA oligonucleotide on a designed biochip. At a concentration of 0.05 µg mL–1, (--catechin gallate and (--gallocatechin gallate showed more than 40% inhibition activity on a nanoparticle-based RNA oligonucleotide biochip system.Keywords: SARS, RNA oligonucleotide, quantum dots, inhibitor, screening

  11. incaRNAfbinv: a web server for the fragment-based design of RNA sequences. (United States)

    Drory Retwitzer, Matan; Reinharz, Vladimir; Ponty, Yann; Waldispühl, Jérôme; Barash, Danny


    In recent years, new methods for computational RNA design have been developed and applied to various problems in synthetic biology and nanotechnology. Lately, there is considerable interest in incorporating essential biological information when solving the inverse RNA folding problem. Correspondingly, RNAfbinv aims at including biologically meaningful constraints and is the only program to-date that performs a fragment-based design of RNA sequences. In doing so it allows the design of sequences that do not necessarily exactly fold into the target, as long as the overall coarse-grained tree graph shape is preserved. Augmented by the weighted sampling algorithm of incaRNAtion, our web server called incaRNAfbinv implements the method devised in RNAfbinv and offers an interactive environment for the inverse folding of RNA using a fragment-based design approach. It takes as input: a target RNA secondary structure; optional sequence and motif constraints; optional target minimum free energy, neutrality and GC content. In addition to the design of synthetic regulatory sequences, it can be used as a pre-processing step for the detection of novel natural occurring RNAs. The two complementary methodologies RNAfbinv and incaRNAtion are merged together and fully implemented in our web server incaRNAfbinv, available at

  12. Improved CTAB Method for Total RNA Extraction of Mature Feicheng Peach Fruit%改良CTAB法提取成熟肥城桃果实的总RNA

    Institute of Scientific and Technical Information of China (English)

    陈长宝; 朱树华; 周杰


    为了从成熟的肥城桃果实中提取纯净完整的RNA,本试验在原CTAB法的基础上进行了改进,并对RNA的完整性、产率和纯度等方面进行评价.结果表明,采用改良CTAB法所得RNA完整性好,条带清晰,无明显降解,28S和18S rRNA的亮度比约为2∶1,A260/A280达1.89,且产率较高,为356.21 μg/g,经RT-PCR后得到一特异条带,表明该RNA可用于后续分子生物学操作.

  13. 肝癌石蜡包埋组织miRNA提取方法的优化%Optimization of MicroRNA extraction method from HCC FFPE tissues

    Institute of Scientific and Technical Information of China (English)

    党裔武; 容敏华; 陈罡


    Objective To modify and optimize the microRNA(miRNA) extraction and detection approach from hepatocellular carcinoma(HCC) formalin fixed paraffin embedded(FFPE) tissues. Methods miRNeasy FFPE kit was performed to investigate the optimizational thickness oi sections from HCC FFPE tissues and. time of K. treatment. Afterwards,expression of miR. 221, miR 146a,let7 a,miR 191,miR 103 and RNU6B was detected by real time RT PCR. The difference of the miRNA expression be tween fresh cells and FFPE tissues from the same cell line was also compared by real time RT PCR. Results 30 μm thickness and 48 h of proteinase K treatment were the best parameters to isolate miRNA. Different expression levels of all the miRNAs could be detected in both the HCC cells samples and clinic FFPE samples. There was no significant variation of the miRNA expression be tween fresh cells and FFPE samples from the same cell lines. Conclusion The optimizational miRNeasy FFPE method is applicable for miRNA extraction from FFPE tissues. The method is productive and can be popularized for the miRNA isolation for different FFPE tissues.%目的 改进并优化从肝癌(HCC)甲醛固定石蜡包埋组织(FFPE)提取并检测微小RNA(miRNA)的方法.方法 使用miRNeasy FFPE试剂盒探讨提取HCC石蜡组织miRNA最佳切片厚度及蛋白酶K作用时间.实时定量RT-PCR检测miR-221、miR-146a、let7-a、miR-191、miR-103及RNU6B的表达量.将HCC细胞制成石蜡组织,对比同一细胞系新鲜细胞及石蜡包埋细胞的各个miRNA表达量差异.结果 切片厚度30 μm,蛋白酶K作用时间48 h时提取miRNA效果最佳.各HCC细胞系及临床HCC石蜡组织均有不同水平的miRNA-221、miR-146a、let7-a、miR-191、miR-103及RNU6B表达.同一细胞系新鲜细胞与石蜡包埋后的各miRNA表达无明显差别.结论 改良后的miRNeasy FFPE 提取方法能提取石蜡组织中的miRNA,可重复性强,可推广用于临床各种石蜡包埋组织的miRNA提取.

  14. Extraction of total RNA from marine invertebrate tissue%海洋无脊椎动物组织总RNA提取方法的探讨

    Institute of Scientific and Technical Information of China (English)

    冯政夫; 王琳; 李文侠; 部凡; 胡彦江; 朱伟


    RNA 提取是分子生物学研究的基础实验,由于海洋无脊椎动物组织结构以及体成分的特殊性,导致现有的 RNA 提取方法不能完全胜任此工作。作者以刺参(Apostichopus japonicus)、栉孔扇贝(Chlamys farreri)和凡纳滨对虾(Litopenaeus vannamei)为研究对象,对异硫氰酸胍RNA提取方法进行了改进。栉孔扇贝和凡纳滨对虾的肝胰腺富含RNase,刺参体壁结构致密、富含胶原蛋白,而成熟期精巢富含多糖,因此在这些动物组织中提取RNA时,在原有方法基础上增加了瞬时匀浆、增加氯仿及酸性酚的抽提次数,或添加高盐溶液等方法。实验结果显示,改进方法后获得的RNA具有完整的28S、18S和5S rRNA条带, A260nm/A280nm和A260nm/A230nm比值分别为1.89~2.0和1.98~2.11,β-actin基因扩增条带清晰,所获得的组织RNA的纯度和质量符合cDNA合成和基因功能研究的要求。%RNA extraction is the basic experiment of the molecular biology study. The particularity of marine in-vertebrate in tissue structure and component results in some disadvantage to obtaining pure RNA using general RNA extraction methods. In this study, sea cucumber, Apostichopus japonicus, scallop, Chlamys farreri and the white shrimp, Litopenaeus vannamei were used as the experimental materials. The method was modified from the method of Guandine Throcyanate Reagent. Hepatopancreas of scallop and white shrimp were rich in endogenous RNase. The sea cucumber body wall was compact and rich in collagen and its testis was rich in polysaccharides. During the process of RNA extraction from these tissues, extraction time of chloroform and phenol-chloroform was increased, and brief homogenate and high concentrated solution of NaCl were used. The results show that A260nm/A280nm and A260nm/A230nm ratio of RNA were 1.89~2.0 and 1.98~2.11, respectively. The bands of 28S, 18S and 5S were integrity, and the amplified band of β-actin gene was clear. The data demonstrate

  15. Towards a “Sample-In, Answer-Out” Point-of-Care Platform for Nucleic Acid Extraction and Amplification: Using an HPV E6/E7 mRNA Model System

    Directory of Open Access Journals (Sweden)

    Anja Gulliksen


    Full Text Available The paper presents the development of a “proof-of-principle” hands-free and self-contained diagnostic platform for detection of human papillomavirus (HPV E6/E7 mRNA in clinical specimens. The automated platform performs chip-based sample preconcentration, nucleic acid extraction, amplification, and real-time fluorescent detection with minimal user interfacing. It consists of two modular prototypes, one for sample preparation and one for amplification and detection; however, a common interface is available to facilitate later integration into one single module. Nucleic acid extracts (n=28 from cervical cytology specimens extracted on the sample preparation chip were tested using the PreTect HPV-Proofer and achieved an overall detection rate for HPV across all dilutions of 50%–85.7%. A subset of 6 clinical samples extracted on the sample preparation chip module was chosen for complete validation on the NASBA chip module. For 4 of the samples, a 100% amplification for HPV 16 or 33 was obtained at the 1 : 10 dilution for microfluidic channels that filled correctly. The modules of a “sample-in, answer-out” diagnostic platform have been demonstrated from clinical sample input through sample preparation, amplification and final detection.

  16. Analysis on the Physicochemical Properties of Ginkgo biloba Leaves after Enzymolysis Based Ultrasound Extraction and Soxhlet Extraction. (United States)

    Zhang, Chang-Wei; Wang, Cheng-Zhang; Tao, Ran


    In this study, high performance liquid chromatography (HPLC), ultraviolet (UV), thermagravimetric analyzer (TGA), pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS), and scanning electron microscope (SEM) were used as measurement techniques, contents of chemical composition, pyrolytic products, thermal stability, morphological characterization of Ginkgo biloba leaves (GBL) acted as the index, and physicochemical properties of GBL after enzymolysis based ultrasound extraction (EBUE) and Soxhlet extraction were studied. The detection results of chemical composition revealed that contents of general flavone, soluble protein, soluble total sugar and protein in the GBL declined significantly after EBUE, and contents of polyprenols and crude fat obviously reduced as well after Soxhlet extraction. Py-GC-MS results indicated that total GC contents of micromolecules with carbon less than 12 from 54.0% before EBUE decline to 8.34% after EBUE. Total GC contents of long-chain fatty acids with carbon less than 20 from 43.0% before EBUE reduced to 27.0% after Soxhlet extraction. Thermal stability results showed that GBL after Soxhlet extraction was easier to decompose than GBL before EBUE. SEM results illustrated that surface structure of GBL was damaged severely after EBUE, compared with GBL before EBUE, while organic solvent extraction had little influence on the morphological characterization of GBL after Soxhlet extraction compared with GBL after EBUE.

  17. Analysis on the Physicochemical Properties of Ginkgo biloba Leaves after Enzymolysis Based Ultrasound Extraction and Soxhlet Extraction

    Directory of Open Access Journals (Sweden)

    Chang-Wei Zhang


    Full Text Available In this study, high performance liquid chromatography (HPLC, ultraviolet (UV, thermagravimetric analyzer (TGA, pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS, and scanning electron microscope (SEM were used as measurement techniques, contents of chemical composition, pyrolytic products, thermal stability, morphological characterization of Ginkgo biloba leaves (GBL acted as the index, and physicochemical properties of GBL after enzymolysis based ultrasound extraction (EBUE and Soxhlet extraction were studied. The detection results of chemical composition revealed that contents of general flavone, soluble protein, soluble total sugar and protein in the GBL declined significantly after EBUE, and contents of polyprenols and crude fat obviously reduced as well after Soxhlet extraction. Py-GC-MS results indicated that total GC contents of micromolecules with carbon less than 12 from 54.0% before EBUE decline to 8.34% after EBUE. Total GC contents of long-chain fatty acids with carbon less than 20 from 43.0% before EBUE reduced to 27.0% after Soxhlet extraction. Thermal stability results showed that GBL after Soxhlet extraction was easier to decompose than GBL before EBUE. SEM results illustrated that surface structure of GBL was damaged severely after EBUE, compared with GBL before EBUE, while organic solvent extraction had little influence on the morphological characterization of GBL after Soxhlet extraction compared with GBL after EBUE.

  18. Inhibition of avian tumor viruses by vector-based RNA interference (United States)

    RNA interference (RNAi) has been shown to reduce the replication of certain animal viruses both in cell culture and in live animals. We developed RNAi-based anti-viral strategies against two important chicken pathogens: avian leukosis virus (ALV) and Marek’s Disease virus MDV). Entry plasmids conta...

  19. Generating Unstable Resonances for Extraction Schemes Based on Transverse Splitting

    CERN Document Server

    Giovannozzi, M; Turchetti, G


    A few years ago, a novel multi-turn extraction scheme was proposed, based on particle trapping inside stable resonances. Numerical simulations and experimental tests have confirmed the feasibility of such a scheme for low order resonances. While the third-order resonance is generically unstable and those higher than fourth-order are generically stable, the fourth-order resonance can be either stable or unstable depending on the specifics of the system under consideration. By means of the Normal Form a general approach to control the stability of the fourth-order resonance has been derived. This approach is based on the control of the amplitude detuning and the general form for a lattice with an arbitrary number of sextupole and octupole families is derived in this paper. Numerical simulations have confirmed the analytical results and have shown that, when crossing the unstable fourth-order resonance, the region around the centre of the phase space is depleted and particles are trapped in only the four stable ...

  20. CEMS using hot wet extractive method based on DOAS (United States)

    Sun, Bo; Zhang, Chi; Sun, Changku


    A continuous emission monitoring system (CEMS) using hot wet extractive method based on differential optical absorption spectroscopy (DOAS) is designed. The developed system is applied to retrieving the concentration of SO2 and NOx in flue gas on-site. The flue gas is carried along a heated sample line into the sample pool at a constant temperature above the dew point. In this case, the adverse impact of water vapor on measurement accuracy is reduced greatly, and the on-line calibration is implemented. And then the flue gas is discharged from the sample pool after the measuring process is complete. The on-site applicability of the system is enhanced by using Programmable Logic Controller (PLC) to control each valve in the system during the measuring and on-line calibration process. The concentration retrieving method used in the system is based on the partial least squares (PLS) regression nonlinear method. The relationship between the known concentration and the differential absorption feature gathered by the PLS nonlinear method can be figured out after the on-line calibration process. Then the concentration measurement of SO2 and NOx can be easily implemented according to the definite relationship. The concentration retrieving method can identify the information and noise effectively, which improves the measuring accuracy of the system. SO2 with four different concentrations are measured by the system under laboratory conditions. The results proved that the full-scale error of this system is less than 2%FS.

  1. PCR-based typing of DNA extracted from cigarette butts. (United States)

    Hochmeister, M N; Budowle, B; Jung, J; Borer, U V; Comey, C T; Dirnhofer, R


    Limited genetic marker information can be obtained from saliva by typing by conventional serological means. Thus, the application of PCR-based DNA typing methods was investigated as a potential approach for typing genetic markers in saliva. DNA was isolated from 200 cigarettes smoked by 10 different individuals (20 cigarettes per individual) and from 3 cigarette butts recovered from 2 crime scenes (adjudicated cases) using a Chelex 100 extraction procedure. The amount of recovered human DNA was quantified by slot-blot analysis and ranged from approximately less than 2-160 ng DNA per cigarette butt for the 200 samples, and 8 ng, 50 ng, and 100 ng for the cigarette butts from the adjudicated cases. The DNA was successfully amplified by the polymerase chain reaction (PCR) for the HLA-DQ alpha locus (99 out of 100 samples) as well as for the variable number of tandem repeat (VNTR) locus D1S80 (99 out of 100 samples). Amplification and typing of DNA was successful on all samples recovered from the crime scenes. The results suggest that PCR-based typing of DNA offers a potential method for genetically characterizing traces of saliva on cigarette butts.

  2. Construction of permanently inducible miRNA-based expression vectors using site-specific recombinases

    Directory of Open Access Journals (Sweden)

    Garwick-Coppens Sara E


    Full Text Available Abstract Background RNA interference (RNAi is a conserved gene silencing mechanism mediated by small inhibitory microRNAs (miRNAs. Promoter-driven miRNA expression vectors have emerged as important tools for delivering natural or artificially designed miRNAs to eukaryotic cells and organisms. Such systems can be used to query the normal or pathogenic functions of natural miRNAs or messenger RNAs, or to therapeutically silence disease genes. Results As with any molecular cloning procedure, building miRNA-based expression constructs requires a time investment and some molecular biology skills. To improve efficiency and accelerate the construction process, we developed a method to rapidly generate miRNA expression vectors using recombinases instead of more traditional cut-and-paste molecular cloning techniques. In addition to streamlining the construction process, our cloning strategy provides vectors with added versatility. In our system, miRNAs can be constitutively expressed from the U6 promoter, or inducibly expressed by Cre recombinase. We also engineered a built-in mechanism to destroy the vector with Flp recombinase, if desired. Finally, to further simplify the construction process, we developed a software package that automates the prediction and design of optimal miRNA sequences using our system. Conclusions We designed and tested a modular system to rapidly clone miRNA expression cassettes. Our strategy reduces the hands-on time required to successfully generate effective constructs, and can be implemented in labs with minimal molecular cloning expertise. This versatile system provides options that permit constitutive or inducible miRNA expression, depending upon the needs of the end user. As such, it has utility for basic or translational applications.

  3. A mRNA-Responsive G-Quadruplex-Based Drug Release System

    Directory of Open Access Journals (Sweden)

    Hidenobu Yaku


    Full Text Available G-quadruplex-based drug delivery carriers (GDDCs were designed to capture and release a telomerase inhibitor in response to a target mRNA. Hybridization between a loop on the GDDC structure and the mRNA should cause the G-quadruplex structure of the GDDC to unfold and release the bound inhibitor, anionic copper(II phthalocyanine (CuAPC. As a proof of concept, GDDCs were designed with a 10-30-mer loop, which can hybridize with a target sequence in epidermal growth factor receptor (EGFR mRNA. Structural analysis using circular dichroism (CD spectroscopy showed that the GDDCs form a (3 + 1 type G-quadruplex structure in 100 mM KCl and 10 mM MgCl2 in the absence of the target RNA. Visible absorbance titration experiments showed that the GDDCs bind to CuAPC with Ka values of 1.5 × 105 to 5.9 × 105 M−1 (Kd values of 6.7 to 1.7 μM at 25 °C, depending on the loop length. Fluorescence titration further showed that the G-quadruplex structure unfolds upon binding to the target RNA with Ka values above 1.0 × 108 M−1 (Kd values below 0.01 μM at 25 °C. These results suggest the carrier can sense and bind to the target RNA, which should result in release of the bound drug. Finally, visible absorbance titration experiments demonstrated that the GDDC release CuAPC in response to the target RNA.

  4. Nanoparticle-based delivery of small interfering RNA: challenges for cancer therapy

    Directory of Open Access Journals (Sweden)

    Miele E


    Full Text Available Evelina Miele,1,* Gian Paolo Spinelli,2,* Ermanno Miele,3 Enzo Di Fabrizio,3,6 Elisabetta Ferretti,4 Silverio Tomao,2 Alberto Gulino,1,5 1Department of Molecular Medicine, 2Department of Medico-Surgical Sciences and Biotechnologies, Sapienza University of Rome, Rome, 3Nanostructures, Istituto Italiano di Tecnologia, via Morego, 30, 16163 Genova, 4Department of Experimental Medicine, Sapienza University of Rome, Rome, 5Center for Life Nanoscience, Istituto Italiano di Tecnologia, Rome, Italy, 6BIONEM lab, University of Magna Graecia, Campus S. Venuta, Viale Europa 88100 Catanzaro, Italy *These authors contributed equally to this workAbstract: During recent decades there have been remarkable advances and profound changes in cancer therapy. Many therapeutic strategies learned at the bench, including monoclonal antibodies and small molecule inhibitors, have been used at the bedside, leading to important successes. One of the most important advances in biology has been the discovery that small interfering RNA (siRNA is able to regulate the expression of genes, by a phenomenon known as RNA interference (RNAi. RNAi is one of the most rapidly growing fields of research in biology and therapeutics. Much research effort has gone into the application of this new discovery in the treatment of various diseases, including cancer. However, even though these molecules may have potential and strong utility, some limitations make their clinical application difficult, including delivery problems, side effects due to off-target actions, disturbance of physiological functions of the cellular machinery involved in gene silencing, and induction of the innate immune response. Many researchers have attempted to overcome these limitations and to improve the safety of potential RNAi-based therapeutics. Nanoparticles, which are nanostructured entities with tunable size, shape, and surface, as well as biological behavior, provide an ideal opportunity to modify current

  5. A plasmonic colorimetric strategy for visual miRNA detection based on hybridization chain reaction (United States)

    Miao, Jie; Wang, Jingsheng; Guo, Jinyang; Gao, Huiguang; Han, Kun; Jiang, Chengmin; Miao, Peng


    In this work, a novel colorimetric strategy for miRNA analysis is proposed based on hybridization chain reaction (HCR)-mediated localized surface plasmon resonance (LSPR) variation of silver nanoparticles (AgNPs). miRNA in the sample to be tested is able to release HCR initiator from a solid interface to AgNPs colloid system by toehold exchange-mediated strand displacement, which then triggers the consumption of fuel strands with single-stranded tails for HCR. The final produced long nicked double-stranded DNA loses the ability to protect AgNPs from salt-induced aggregation. The stability variation of the colloid system can then be monitored by recording corresponding UV-vis spectrum and initial miRNA level is thus determined. This sensing system involves only four DNA strands which is quite simple. The practical utility is confirmed to be excellent by employing different biological samples.

  6. An siRNA-based method for efficient silencing of gene expression in mature brown adipocytes. (United States)

    Isidor, Marie S; Winther, Sally; Basse, Astrid L; Petersen, M Christine H; Cannon, Barbara; Nedergaard, Jan; Hansen, Jacob B


    Brown adipose tissue is a promising therapeutic target for opposing obesity, glucose intolerance and insulin resistance. The ability to modulate gene expression in mature brown adipocytes is important to understand brown adipocyte function and delineate novel regulatory mechanisms of non-shivering thermogenesis. The aim of this study was to optimize a lipofection-based small interfering RNA (siRNA) transfection protocol for efficient silencing of gene expression in mature brown adipocytes. We determined that a critical parameter was to deliver the siRNA to mature adipocytes by reverse transfection, i.e. transfection of non-adherent cells. Using this protocol, we effectively knocked down both high- and low-abundance transcripts in a model of mature brown adipocytes (WT-1) as well as in primary mature mouse brown adipocytes. A functional consequence of the knockdown was confirmed by an attenuated increase in uncoupled respiration (thermogenesis) in response to β-adrenergic stimulation of mature WT-1 brown adipocytes transfected with uncoupling protein 1 siRNA. Efficient gene silencing was also obtained in various mouse and human white adipocyte models (3T3-L1, primary mouse white adipocytes, hMADS) with the ability to undergo "browning." In summary, we report an easy and versatile reverse siRNA transfection protocol to achieve specific silencing of gene expression in various models of mature brown and browning-competent white adipocytes, including primary cells.

  7. An accurate assay for HCV based on real-time fluorescence detection of isothermal RNA amplification. (United States)

    Wu, Xuping; Wang, Jianfang; Song, Jinyun; Li, Jiayan; Yang, Yongfeng


    Hepatitis C virus (HCV) is one of the common reasons of liver fibrosis and hepatocellular carcinoma (HCC). Early, rapid and accurate HCV RNA detection is important to prevent and control liver disease. A simultaneous amplification and testing (SAT) assay, which is based on isothermal amplification of RNA and real-time fluorescence detection, was designed to optimize routine HCV RNA detection. In this study, HCV RNA and an internal control (IC) were amplified and analyzed simultaneously by SAT assay and detection of fluorescence using routine real-time PCR equipment. The assay detected as few as 10 copies of HCV RNA transcripts. We tested 705 serum samples with SAT, among which 96.4% (680/705) showed consistent results compared with routine real-time PCR. About 92% (23/25) discordant samples were confirmed to be same results as SAT-HCV by using a second real-time PCR. The sensitivity and specificity of SAT-HCV assay were 99.6% (461/463) and 100% (242/242), respectively. In conclusion, the SAT assay is an accurate test with a high specificity and sensitivity which may increase the detection rate of HCV. It is therefore a promising tool to diagnose HCV infection.

  8. Novel lipoproteoplex delivers Keap1 siRNA based gene therapy to accelerate diabetic wound healing. (United States)

    Rabbani, Piul S; Zhou, Anna; Borab, Zachary M; Frezzo, Joseph A; Srivastava, Nikita; More, Haresh T; Rifkin, William J; David, Joshua A; Berens, Samuel J; Chen, Raymond; Hameedi, Sophia; Junejo, Muhammad H; Kim, Camille; Sartor, Rita A; Liu, Che F; Saadeh, Pierre B; Montclare, Jin K; Ceradini, Daniel J


    Therapeutics utilizing siRNA are currently limited by the availability of safe and effective delivery systems. Cutaneous diseases, specifically ones with significant genetic components are ideal candidates for topical siRNA based therapy but the anatomical structure of skin presents a considerable hurdle. Here, we optimized a novel liposome and protein hybrid nanoparticle delivery system for the topical treatment of diabetic wounds with severe oxidative stress. We utilized a cationic lipid nanoparticle (CLN) composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the edge activator sodium cholate (NaChol), in a 6:1 ratio of DOTAP:NaChol (DNC). Addition of a cationic engineered supercharged coiled-coil protein (CSP) in a 10:1:1 ratio of DNC:CSP:siRNA produced a stable lipoproteoplex (LPP) nanoparticle, with optimal siRNA complexation, minimal cytotoxicity, and increased transfection efficacy. In a humanized murine diabetic wound healing model, our optimized LPP formulation successfully delivered siRNA targeted against Keap1, key repressor of Nrf2 which is a central regulator of redox mechanisms. Application of LPP complexing siKeap1 restored Nrf2 antioxidant function, accelerated diabetic tissue regeneration, and augmented reduction-oxidation homeostasis in the wound environment. Our topical LPP delivery system can readily be translated into clinical use for the treatment of diabetic wounds and can be extended to other cutaneous diseases with genetic components.

  9. An RNA-based analysis of changes in biodiversity indices in response to Sus scrofa domesticus decomposition. (United States)

    Bergmann, R C; Ralebitso-Senior, T K; Thompson, T J U


    Despite emergent research initiatives, significant knowledge gaps remain of soil microbiology-associated cadaver decomposition. Nevertheless, preliminary studies have shown that the vast diversity and complex interactions of soil microbial communities have great potential for forensic applications such as clandestine grave location and postmortem interval estimation. This study investigated changes in soil bacterial communities during pig (Sus scrofa domesticus) leg decomposition. 16S rRNA, instead of the usually applied 16S rDNA marker, was used to compare the metabolically active bacteria. Total bacterial RNA was extracted from soil samples of three different layers on day 3, 28 and 77 after the shallow burial of a pig leg. The V3 region of the 16S rRNA was amplified, analysed by RT-PCR DGGE, and compared with control soil bacterial community profiles. Statistically significant differences in soil bacterial biodiversity were observed. For the control, bacterial diversity (H') and species richness (S) of the three layers averaged 2.48±0.14 (H') and 18.8±2.5 (S), respectively, while for the test soil increases (p=0.027) were recorded between day 3 (H'=2.71±0.02; S=21.3±2.0) and 28 (H'=3.46±0.32; S=60.3±16.9), particularly in the middle (10-20 cm) and bottom (20-30 cm) soil layers. Between day 28 and 77 the diversity and richness then decreased on average for all three layers (H'=3.43±0.20; S=60.0±17.3) but remained higher than on day 3. Thus, responses in soil bacterial profiles and activity to carcass decomposition, detected and characterised by RNA-based DGGE, could be used together with RNA sequencing data, changes in physico-chemical variables (carbon, nitrogen, phosphorus, temperature, redox potential, water activity and pH) and conventional macroecology markers (e.g. insects and vegetation), to develop a suite of analytical protocols for different forensic scenarios.

  10. A rapid and versatile combined DNA/RNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsis thaliana ecotypes Col-0 and Landsberg erecta

    Directory of Open Access Journals (Sweden)

    Coupland George


    Full Text Available Abstract Background Many established PCR-based approaches in plant molecular biology rely on lengthy and expensive methods for isolation of nucleic acids. Although several rapid DNA isolation protocols are available, they have not been tested for simultaneous RNA isolation for RT-PCR applications. In addition, traditional map-based cloning technologies often use ill-proportioned marker regions even when working with the model plant Arabidopsis thaliana, where the availability of the full genome sequence can now be exploited for the creation of a high-density marker systems. Results We designed a high-density polymorphic marker set between two frequently used ecotypes. This new polymorphic marker set allows size separation of PCR products on agarose gels and provides an initial resolution of 10 cM in linkage mapping experiments, facilitated by a rapid plant nucleic acid extraction protocol using minimal amounts of A. thaliana tissue. Using this extraction protocol, we have also characterized segregating T-DNA insertion mutations. In addition, we have shown that our rapid nucleic acid extraction protocol can also be used for monitoring transcript levels by RT-PCR amplification. Finally we have demonstrated that our nucleic acid isolation method is also suitable for other plant species, such as tobacco and barley. Conclusion To facilitate high-throughput linkage mapping and other genomic applications, our nucleic acid isolation protocol yields sufficient quality of DNA and RNA templates for PCR and RT-PCR reactions, respectively. This new technique requires considerably less time compared to other purification methods, and in combination with a new polymorphic PCR marker set dramatically reduces the workload required for linkage mapping of mutations in A. thaliana utilizing crosses between Col-0 and Landsberg erecta (Ler ecotypes.

  11. Extraction of alkaloids for NMR-based profiling

    DEFF Research Database (Denmark)

    Yilmaz, Ali; Nyberg, Nils; Jaroszewski, Jerzy W.


    A museum collection of Cinchona cortex samples (n = 117), from the period 1850–1950, were extracted with a mixture of chloroform-d1, methanol-d4, water-d2, and perchloric acid in the ratios 5:5:1:1. The extracts were directly analyzed using 1H NMR spectroscopy (600 MHz) and the spectra evaluated ...

  12. BEaST: brain extraction based on nonlocal segmentation technique.

    NARCIS (Netherlands)

    Eskildsen, S.F.; Coupe, P.; Fonov, V.; Manjon, J.V.; Leung, K.K.; Guizard, N.; Wassef, S.N.; Ostergaard, L.R.; Collins, D.L.; Olde Rikkert, M.


    Brain extraction is an important step in the analysis of brain images. The variability in brain morphology and the difference in intensity characteristics due to imaging sequences make the development of a general purpose brain extraction algorithm challenging. To address this issue, we propose a ne

  13. Apriori and N-gram Based Chinese Text Feature Extraction Method

    Institute of Scientific and Technical Information of China (English)

    王晔; 黄上腾


    A feature extraction, which means extracting the representative words from a text, is an important issue in text mining field. This paper presented a new Apriori and N-gram based Chinese text feature extraction method, and analyzed its correctness and performance. Our method solves the question that the exist extraction methods cannot find the frequent words with arbitrary length in Chinese texts. The experimental results show this method is feasible.

  14. Single-point mutation detection in RNA extracts using gold nanoparticles modified with hydrophobic molecular beacon-like structures. (United States)

    Latorre, Alfonso; Posch, Christian; Garcimartín, Yolanda; Ortiz-Urda, Susana; Somoza, Álvaro


    Gold nanoparticles functionalized with oligonucleotides that bear a cholesterol group are used as gene sensors. The hydrophobic molecule is buried inside the nanostructure but when the complementary RNA sequence is present the structure unfolds exposing the cholesterol group to the water molecules. This rearrangement leads to the aggregation of the nanostructures.

  15. Classification of Textures Using Filter Based Local Feature Extraction

    Directory of Open Access Journals (Sweden)

    Bocekci Veysel Gokhan


    Full Text Available In this work local features are used in feature extraction process in image processing for textures. The local binary pattern feature extraction method from textures are introduced. Filtering is also used during the feature extraction process for getting discriminative features. To show the effectiveness of the algorithm before the extraction process, three different noise are added to both train and test images. Wiener filter and median filter are used to remove the noise from images. We evaluate the performance of the method with Naïve Bayesian classifier. We conduct the comparative analysis on benchmark dataset with different filtering and size. Our experiments demonstrate that feature extraction process combine with filtering give promising results on noisy images.

  16. A knowledge-based scoring function for protein-RNA interactions derived from a statistical mechanics-based iterative method. (United States)

    Huang, Sheng-You; Zou, Xiaoqin


    Protein-RNA interactions play important roles in many biological processes. Given the high cost and technique difficulties in experimental methods, computationally predicting the binding complexes from individual protein and RNA structures is pressingly needed, in which a reliable scoring function is one of the critical components. Here, we have developed a knowledge-based scoring function, referred to as ITScore-PR, for protein-RNA binding mode prediction by using a statistical mechanics-based iterative method. The pairwise distance-dependent atomic interaction potentials of ITScore-PR were derived from experimentally determined protein-RNA complex structures. For validation, we have compared ITScore-PR with 10 other scoring methods on four diverse test sets. For bound docking, ITScore-PR achieved a success rate of up to 86% if the top prediction was considered and up to 94% if the top 10 predictions were considered, respectively. For truly unbound docking, the respective success rates of ITScore-PR were up to 24 and 46%. ITScore-PR can be used stand-alone or easily implemented in other docking programs for protein-RNA recognition.

  17. Morphological operation based dense houses extraction from DSM (United States)

    Li, Y.; Zhu, L.; Tachibana, K.; Shimamura, H.


    This paper presents a method of reshaping and extraction of markers and masks of the dense houses from the DSM based on mathematical morphology (MM). Houses in a digital surface model (DSM) are almost joined together in high-density housing areas, and most segmentation methods cannot completely separate them. We propose to label the markers of the buildings firstly and segment them into masks by watershed then. To avoid detecting more than one marker for a house or no marker at all due to its higher neighbour, the DSM is morphologically reshaped. It is carried out by a MM operation using the certain disk shape SE of the similar size to the houses. The sizes of the houses need to be estimated before reshaping. A granulometry generated by opening-by-reconstruction to the NDSM is proposed to detect the scales of the off-terrain objects. It is a histogram of the global volume of the top hats of the convex objects in the continuous scales. The obvious step change in the profile means that there are many objects of similar sizes occur at this scale. In reshaping procedure, the slices of the object are derived by morphological filtering at the detected continuous scales and reconstructed in pile as the dome. The markers are detected on the basis of the domes.


    Institute of Scientific and Technical Information of China (English)

    LI Jiangxiong; KE Yinglin; LI An; ZHU Weidong


    A principal direction Gaussian image (PDGI)-based algorithm is proposed to extract the regular swept surface from point cloud. Firstly, the PDGI of the regular swept surface is constructed from point cloud, then the bounding box of the Gaussian sphere is uniformly partitioned into a number of small cubes (3D grids) and the PDGI points on the Gaussian sphere are associated with the corresponding 3D grids. Secondly, cluster analysis technique is used to sort out a group of 3D grids containing more PDGI points among the 3D grids. By the connected-region growing algorithm, the congregation point or the great circle is detected from the 3D grids. Thus the translational direction is determined by the congregation point and the direction of the rotational axis is determined by the great circle. In addition, the positional point of the rotational axis is obtained by the intersection of all the projected normal lines of the rotational surface on the plane being perpendicular to the estimated direction of the rotational axis. Finally, a pattern search method is applied to optimize the translational direction and the rotational axis. Some experiments are used to illustrate the feasibility of the above algorithm.

  19. RNA degradation compromises the reliability of microRNA expression profiling

    Directory of Open Access Journals (Sweden)

    Muckenthaler Martina U


    Full Text Available Abstract Background MicroRNAs are small non-coding RNAs that post-transcriptionally regulate gene expression and their expression is frequently altered in human diseases, including cancer. To correlate clinically relevant parameters with microRNA expression, total RNA is frequently prepared from samples that were archived for various time periods in frozen tissue banks but, unfortunately, RNA integrity is not always preserved in these frozen tissues. Here, we investigate whether experimentally induced RNA degradation affects microRNA expression profiles. Results Tissue samples were maintained on ice for defined time periods prior to total RNA extraction, which resulted in different degrees of RNA degradation. MicroRNA expression was then analyzed by microarray analysis (miCHIP or microRNA-specific real-time quantitative PCR (miQPCR. Our results demonstrate that the loss of RNA integrity leads to in unpredictability of microRNA expression profiles for both, array-based and miQPCR assays. Conclusion MicroRNA expression cannot be reliably profiled in degraded total RNA. For the profiling of microRNAs we recommend use of RNA samples with a RNA integrity number equal to or above seven.

  20. A reliable and inexpensive method of nucleic acid extraction for the PCR-based detection of diverse plant pathogens. (United States)

    Li, R; Mock, R; Huang, Q; Abad, J; Hartung, J; Kinard, G


    A reliable extraction method is described for the preparation of total nucleic acids from at least ten plant genera for subsequent detection of plant pathogens by PCR-based techniques. The method combined a modified CTAB (cetyltrimethylammonium bromide) extraction protocol with a semi-automatic homogenizer (FastPrep) instrument) for rapid sample processing and low potential of cross contamination. The method was applied to sample preparation for PCR-based detection of 28 different RNA and DNA viruses, six viroids, two phytoplasmas and two bacterial pathogens from a range of infected host plants including sweet potato, small fruits and fruit trees. The procedure is cost-effective and the qualities of the nucleic acid preparations are comparable to those prepared by commonly used commercial kits. The efficiency of the procedure permits processing of numerous samples and the use of a single nucleic acid preparation for testing both RNA and DNA genomes by PCR, making this an appealing method for testing multiple pathogens in certification and quarantine programs.

  1. A quantitative analysis of secondary RNA structure using domination based parameters on trees

    Directory of Open Access Journals (Sweden)

    Zou Yue


    Full Text Available Abstract Background It has become increasingly apparent that a comprehensive database of RNA motifs is essential in order to achieve new goals in genomic and proteomic research. Secondary RNA structures have frequently been represented by various modeling methods as graph-theoretic trees. Using graph theory as a modeling tool allows the vast resources of graphical invariants to be utilized to numerically identify secondary RNA motifs. The domination number of a graph is a graphical invariant that is sensitive to even a slight change in the structure of a tree. The invariants selected in this study are variations of the domination number of a graph. These graphical invariants are partitioned into two classes, and we define two parameters based on each of these classes. These parameters are calculated for all small order trees and a statistical analysis of the resulting data is conducted to determine if the values of these parameters can be utilized to identify which trees of orders seven and eight are RNA-like in structure. Results The statistical analysis shows that the domination based parameters correctly distinguish between the trees that represent native structures and those that are not likely candidates to represent RNA. Some of the trees previously identified as candidate structures are found to be "very" RNA like, while others are not, thereby refining the space of structures likely to be found as representing secondary RNA structure. Conclusion Search algorithms are available that mine nucleotide sequence databases. However, the number of motifs identified can be quite large, making a further search for similar motif computationally difficult. Much of the work in the bioinformatics arena is toward the development of better algorithms to address the computational problem. This work, on the other hand, uses mathematical descriptors to more clearly characterize the RNA motifs and thereby reduce the corresponding search space. These

  2. Switch to raltegravir-based regimens and HIV DNA decrease in patients with suppressed HIV RNA

    Directory of Open Access Journals (Sweden)

    Claudia Bianco


    Full Text Available Introduction: Raltegravir intensification is associated with an increase in 2-LTR episomal HIV DNA= circles, indicating a persistent low-level replication, in some individuals in ART with suppressed HIV RNA. We aimed at monitoring residual plasma HIV RNA and cellular HIV DNA in virologically suppressed patients switching to a raltegravir-based regimen. Materials and Methods: Forty-six HIV-infected subjects on PI or NNRTI based-regimens, with plasma HIV RNA level 200 cells/µL for ≥12 months were enrolled. Thirty-four patients switched to raltegravir-based regimen (RASTA study group and 12 continued a PI or NNRTI based-regimen (control group. Ultrasensitive HIV residual viremia and total PBMC HIV DNA were assessed at baseline (W0, 24 (W24 and 48 (W48 weeks. HIV RNA levels were determined by an ultrasensitive test derived from a commercial real time PCR (limit of detection 5 copies/ml. A real time PCR was used to quantify HIV DNA copy numbers in PBMCs. Results: At W0, HIV DNA was detected in all patients while at W48 it was detectable in 82.3% of RASTA group vs 100% of controls (p=0.01. The difference between the average values of HIV DNA log10 copies/10°6 CD4 at W0 (median 3.11, IQR 2.70–3.45 and W48 (median 2.87, IQR 2.24–3.38 was statistically significant for RASTA group (p=0.035. Male gender (mean difference −0.37 log10 copies/10°6 PBMC, p=0.023 and previous PI based-ART (mean difference +0.39 log10 copies/10°6 PBMC, p=0.036 were predictive of HIV DNA level at W0. After adjusting for previous PI based-ART, male gender was the only variable independently associated with HIV DNA size at W0 (mean difference −0.326 log10 copies/10°6 PBMC, 95% CI −0.641, −0.011 p=0.043. Ultrasensitive HIV-1 RNA was detectable at W0 in 50% of RASTA group versus 66.7% of controls and at W48 in 32.4% versus 45.5%, respectively. No differences were found between HIV RNA levels at W0 and W48 within and between the two groups. Conclusions: Switching to

  3. An improved approach for flow-based cloud point extraction. (United States)

    Frizzarin, Rejane M; Rocha, Fábio R P


    Novel strategies are proposed to circumvent the main drawbacks of flow-based cloud point extraction (CPE). The surfactant-rich phase (SRP) was directly retained into the optical path of the spectrophotometric cell, thus avoiding its dilution previously to the measurement and yielding higher sensitivity. Solenoid micro-pumps were exploited to improve mixing by the pulsed flow and also to modulate the flow-rate for retention and removal of the SRP, thus avoiding the elution step, often carried out with organic solvents. The heat released and the increase of the salt concentration provided by an on-line neutralization reaction were exploited to induce the cloud point without an external heating device. These innovations were demonstrated by the spectrophotometric determination of iron, yielding a linear response from 10 to 200 μg L(-1) with a coefficient of variation of 2.3% (n=7). Detection limit and sampling rate were estimated at 5 μg L(-1) (95% confidence level) and 26 samples per hour, respectively. The enrichment factor was 8.9 and the procedure consumed only 6 μg of TAN and 390 μg of Triton X-114 per determination. At the 95% confidence level, the results obtained for freshwater samples agreed with the reference procedure and those obtained for digests of bovine muscle, rice flour, brown bread and tort lobster agreed with the certified reference values. The proposed procedure thus shows advantages in relation to previously proposed approaches for flow-based CPE, being a fast and environmental friendly alternative for on-line separation and pre-concentration.

  4. Polyphenolic extracts from cowpea (Vigna unguiculata) protect colonic myofibroblasts (CCD18Co cells) from lipopolysaccharide (LPS)-induced inflammation--modulation of microRNA 126. (United States)

    Ojwang, Leonnard O; Banerjee, Nivedita; Noratto, Giuliana D; Angel-Morales, Gabriela; Hachibamba, Twambo; Awika, Joseph M; Mertens-Talcott, Susanne U


    Cowpea (Vigna unguiculata) is a drought tolerant crop with several agronomic advantages over other legumes. This study evaluated varieties from four major cowpea phenotypes (black, red, light brown and white) containing different phenolic profiles for their anti-inflammatory property on non-malignant colonic myofibroblasts (CCD18Co) cells challenged with an endotoxin (lipopolysaccharide, LPS). Intracellular reactive oxygen species (ROS) assay on the LPS-stimulated cells revealed antioxidative potential of black and red cowpea varieties. Real-time qRT-PCR analysis in LPS-stimulated cells revealed down-regulation of proinflammatory cytokines (IL-8, TNF-α, VCAM-1), transcription factor NF-κB and modulation of microRNA-126 (specific post-transcriptional regulator of VCAM-1) by cowpea polyphenolics. The ability of cowpea polyphenols to modulate miR-126 signaling and its target gene VCAM-1 were studied in LPS-stimulated endothelial cells transfected with a specific inhibitor of miR-126, and treated with 10 mg GAE/L black cowpea extract where the extract in part reversed the effect of the miR-126 inhibitor. This suggests that cowpea may exert their anti-inflammatory activities at least in part through induction of miR-126 that then down-regulate VCAM-1 mRNA and protein expressions. Overall, Cowpea therefore is promising as an anti-inflammatory dietary component.

  5. RAID: a comprehensive resource for human RNA-associated (RNA-RNA/RNA-protein) interaction. (United States)

    Zhang, Xiaomeng; Wu, Deng; Chen, Liqun; Li, Xiang; Yang, Jinxurong; Fan, Dandan; Dong, Tingting; Liu, Mingyue; Tan, Puwen; Xu, Jintian; Yi, Ying; Wang, Yuting; Zou, Hua; Hu, Yongfei; Fan, Kaili; Kang, Juanjuan; Huang, Yan; Miao, Zhengqiang; Bi, Miaoman; Jin, Nana; Li, Kongning; Li, Xia; Xu, Jianzhen; Wang, Dong


    Transcriptomic analyses have revealed an unexpected complexity in the eukaryote transcriptome, which includes not only protein-coding transcripts but also an expanding catalog of noncoding RNAs (ncRNAs). Diverse coding and noncoding RNAs (ncRNAs) perform functions through interaction with each other in various cellular processes. In this project, we have developed RAID (, an RNA-associated (RNA-RNA/RNA-protein) interaction database. RAID intends to provide the scientific community with all-in-one resources for efficient browsing and extraction of the RNA-associated interactions in human. This version of RAID contains more than 6100 RNA-associated interactions obtained by manually reviewing more than 2100 published papers, including 4493 RNA-RNA interactions and 1619 RNA-protein interactions. Each entry contains detailed information on an RNA-associated interaction, including RAID ID, RNA/protein symbol, RNA/protein categories, validated method, expressing tissue, literature references (Pubmed IDs), and detailed functional description. Users can query, browse, analyze, and manipulate RNA-associated (RNA-RNA/RNA-protein) interaction. RAID provides a comprehensive resource of human RNA-associated (RNA-RNA/RNA-protein) interaction network. Furthermore, this resource will help in uncovering the generic organizing principles of cellular function network.

  6. Chemiluminescence imaging for microRNA detection based on cascade exponential isothermal amplification machinery. (United States)

    Xu, Yongjie; Li, Dandan; Cheng, Wei; Hu, Rong; Sang, Ye; Yin, Yibing; Ding, Shijia; Ju, Huangxian


    A novel G-quadruplex DNAzyme-driven chemiluminescence (CL) imaging method was developed for ultrasensitive and specific detection of miRNA based on the cascade exponential isothermal amplification reaction (EXPAR) machinery. A structurally tailored hairpin probe switch was designed to selectively recognise miRNA and form hybridisation products to trigger polymerase and nicking enzyme machinery, resulting in the generation of product I, which was complementary to a region of the functional linear template. Then, the response of the functional linear template to the generated product I further activated the exponential isothermal amplification machinery, leading to synthesis of numerous horseradish peroxidase mimicking DNAzyme units for CL signal transduction. The amplification paradigm generated a linear response from 10 fM to 100 pM, with a low detection limit of 2.91 fM, and enabled discrimination of target miRNA from a single-base mismatched target. The developed biosensing platform demonstrated the advantages of isothermal, homogeneous, visual detection for miRNA assays, offering a promising tool for clinical diagnosis.

  7. Differential expression analysis of RNA-seq data at single-base resolution. (United States)

    Frazee, Alyssa C; Sabunciyan, Sarven; Hansen, Kasper D; Irizarry, Rafael A; Leek, Jeffrey T


    RNA-sequencing (RNA-seq) is a flexible technology for measuring genome-wide expression that is rapidly replacing microarrays as costs become comparable. Current differential expression analysis methods for RNA-seq data fall into two broad classes: (1) methods that quantify expression within the boundaries of genes previously published in databases and (2) methods that attempt to reconstruct full length RNA transcripts. The first class cannot discover differential expression outside of previously known genes. While the second approach does possess discovery capabilities, statistical analysis of differential expression is complicated by the ambiguity and variability incurred while assembling transcripts and estimating their abundances. Here, we propose a novel method that first identifies differentially expressed regions (DERs) of interest by assessing differential expression at each base of the genome. The method then segments the genome into regions comprised of bases showing similar differential expression signal, and then assigns a measure of statistical significance to each region. Optionally, DERs can be annotated using a reference database of genomic features. We compare our approach with leading competitors from both current classes of differential expression methods and highlight the strengths and weaknesses of each. A software implementation of our method is available on github (

  8. Covariant Evolutionary Event Analysis for Base Interaction Prediction Using a Relational Database Management System for RNA. (United States)

    Xu, Weijia; Ozer, Stuart; Gutell, Robin R


    With an increasingly large amount of sequences properly aligned, comparative sequence analysis can accurately identify not only common structures formed by standard base pairing but also new types of structural elements and constraints. However, traditional methods are too computationally expensive to perform well on large scale alignment and less effective with the sequences from diversified phylogenetic classifications. We propose a new approach that utilizes coevolutional rates among pairs of nucleotide positions using phylogenetic and evolutionary relationships of the organisms of aligned sequences. With a novel data schema to manage relevant information within a relational database, our method, implemented with a Microsoft SQL Server 2005, showed 90% sensitivity in identifying base pair interactions among 16S ribosomal RNA sequences from Bacteria, at a scale 40 times bigger and 50% better sensitivity than a previous study. The results also indicated covariation signals for a few sets of cross-strand base stacking pairs in secondary structure helices, and other subtle constraints in the RNA structure.

  9. Bias in ligation-based small RNA sequencing library construction is determined by adaptor and RNA structure.

    Directory of Open Access Journals (Sweden)

    Ryan T Fuchs

    Full Text Available High-throughput sequencing (HTS has become a powerful tool for the detection of and sequence characterization of microRNAs (miRNA and other small RNAs (sRNA. Unfortunately, the use of HTS data to determine the relative quantity of different miRNAs in a sample has been shown to be inconsistent with quantitative PCR and Northern Blot results. Several recent studies have concluded that the major contributor to this inconsistency is bias introduced during the construction of sRNA libraries for HTS and that the bias is primarily derived from the adaptor ligation steps, specifically where single stranded adaptors are sequentially ligated to the 3' and 5'-end of sRNAs using T4 RNA ligases. In this study we investigated the effects of ligation bias by using a pool of randomized ligation substrates, defined mixtures of miRNA sequences and several combinations of adaptors in HTS library construction. We show that like the 3' adaptor ligation step, the 5' adaptor ligation is also biased, not because of primary sequence, but instead due to secondary structures of the two ligation substrates. We find that multiple secondary structural factors influence final representation in HTS results. Our results provide insight about the nature of ligation bias and allowed us to design adaptors that reduce ligation bias and produce HTS results that more accurately reflect the actual concentrations of miRNAs in the defined starting material.

  10. Identification of threats using linguistics-based knowledge extraction.

    Energy Technology Data Exchange (ETDEWEB)

    Chew, Peter A.


    One of the challenges increasingly facing intelligence analysts, along with professionals in many other fields, is the vast amount of data which needs to be reviewed and converted into meaningful information, and ultimately into rational, wise decisions by policy makers. The advent of the world wide web (WWW) has magnified this challenge. A key hypothesis which has guided us is that threats come from ideas (or ideology), and ideas are almost always put into writing before the threats materialize. While in the past the 'writing' might have taken the form of pamphlets or books, today's medium of choice is the WWW, precisely because it is a decentralized, flexible, and low-cost method of reaching a wide audience. However, a factor which complicates matters for the analyst is that material published on the WWW may be in any of a large number of languages. In 'Identification of Threats Using Linguistics-Based Knowledge Extraction', we have sought to use Latent Semantic Analysis (LSA) and other similar text analysis techniques to map documents from the WWW, in whatever language they were originally written, to a common language-independent vector-based representation. This then opens up a number of possibilities. First, similar documents can be found across language boundaries. Secondly, a set of documents in multiple languages can be visualized in a graphical representation. These alone offer potentially useful tools and capabilities to the intelligence analyst whose knowledge of foreign languages may be limited. Finally, we can test the over-arching hypothesis--that ideology, and more specifically ideology which represents a threat, can be detected solely from the words which express the ideology--by using the vector-based representation of documents to predict additional features (such as the ideology) within a framework based on supervised learning. In this report, we present the results of a three-year project of the same name. We believe

  11. Modified RNA extraction from field woody plants for the routine detection of PDV and PNRSV in cherry by RT-PCR%改良RNA提取法及樱桃PDV和PNRSV的RT-PCR检测

    Institute of Scientific and Technical Information of China (English)

    阮小凤; Jelkmann Wilhelm; 马锋旺


    介绍了一种从木本植物组织中获得高质量RNA的快速、简单和高效的核酸提取方法.该方法是基于核酸的二氧化硅捕获,避免了使用苯酚、氯仿等有机溶剂.利用该方法从樱桃组织中提取的总RNA用RT-PCR技术检测PDV,PNRSV均获得成功.从感病植株的一年生枝的叶片、韧皮部及芽组织中扩增出了预期的目的片段,即172和449 bp,而健康组织中无此扩增带.该法提取的总RNA用于RT-PCR技术检测,其敏感性至少与商业出售的Qiagen RNeasy提取试剂盒相当,但简单经济.%An efficient and effective procedure for the extraction of high-quality RNA from woody plants without the use of phenol,organic solvents,or alcohol precipitation is described,which is based on silica capture.The method described has been successfully used for the detection of PDV and PNRSV in cherry by RT-PCR assay using DNA primers for the viral coat protein region.The expected sizes of the amplified products were 172 and 449 bp.Samples from bark,leaves and buds were used.Detection of viral RNA in samples of total plant RNA prepared using this method was found to be as sensitive as the methods previously described using the commercially available Qiagen's RNeasy extraction kit.

  12. Purification of form AI and AII DNA-dependent RNA polymerases from rat-liver nucleoli using low-ionic-strength extraction conditions. (United States)

    Coupar, B E; Chesterton, C J


    Recent findings have confirmed the role of form A DNA-dependent polymerase activity as that which is responsible for the transcription of the ribosomal RNA-coding genes. Unfortunately, the form A enzymes have proved to be very labile and difficult to work with, especially under high ionic strength conditions. We have, therefore, investigated a method for the purification of the form AI and AII enzymes from rat liver using mild low-ionic-strength conditions. Since preparations from whole nuclei were found to be grossly contaminated with protein having similar properties, the enzymes are extracted from nucleoli. Forms AI and AII are separated on a phosphocellulose column, purified by further ion-exchange chromatography, and by sedimentation through a glycerol gradient. The purified enzymes each migrate as a single band on native polyacrylamide gels and have the expected characteristics of form A RNA polymerase. Sedimentation rates through glycerol gradients indicate that they both have a similar size to that of Escherichia coli RNA polymerase (Mr about 500,000). The purified enzymes are free of DNase and RNase. A method is also described for the purification of form B from the nucleoplasm remaining after isolation of nucleoli. The presence of form C activity was not detected.

  13. Taxonomic resolutions based on 18S rRNA genes: a case study of subclass copepoda.

    Directory of Open Access Journals (Sweden)

    Shu Wu

    Full Text Available Biodiversity studies are commonly conducted using 18S rRNA genes. In this study, we compared the inter-species divergence of variable regions (V1-9 within the copepod 18S rRNA gene, and tested their taxonomic resolutions at different taxonomic levels. Our results indicate that the 18S rRNA gene is a good molecular marker for the study of copepod biodiversity, and our conclusions are as follows: 1 18S rRNA genes are highly conserved intra-species (intra-species similarities are close to 100%; and could aid in species-level analyses, but with some limitations; 2 nearly-whole-length sequences and some partial regions (around V2, V4, and V9 of the 18S rRNA gene can be used to discriminate between samples at both the family and order levels (with a success rate of about 80%; 3 compared with other regions, V9 has a higher resolution at the genus level (with an identification success rate of about 80%; and 4 V7 is most divergent in length, and would be a good candidate marker for the phylogenetic study of Acartia species. This study also evaluated the correlation between similarity thresholds and the accuracy of using nuclear 18S rRNA genes for the classification of organisms in the subclass Copepoda. We suggest that sample identification accuracy should be considered when a molecular sequence divergence threshold is used for taxonomic identification, and that the lowest similarity threshold should be determined based on a pre-designated level of acceptable accuracy.

  14. Taxonomic resolutions based on 18S rRNA genes: a case study of subclass copepoda. (United States)

    Wu, Shu; Xiong, Jie; Yu, Yuhe


    Biodiversity studies are commonly conducted using 18S rRNA genes. In this study, we compared the inter-species divergence of variable regions (V1-9) within the copepod 18S rRNA gene, and tested their taxonomic resolutions at different taxonomic levels. Our results indicate that the 18S rRNA gene is a good molecular marker for the study of copepod biodiversity, and our conclusions are as follows: 1) 18S rRNA genes are highly conserved intra-species (intra-species similarities are close to 100%); and could aid in species-level analyses, but with some limitations; 2) nearly-whole-length sequences and some partial regions (around V2, V4, and V9) of the 18S rRNA gene can be used to discriminate between samples at both the family and order levels (with a success rate of about 80%); 3) compared with other regions, V9 has a higher resolution at the genus level (with an identification success rate of about 80%); and 4) V7 is most divergent in length, and would be a good candidate marker for the phylogenetic study of Acartia species. This study also evaluated the correlation between similarity thresholds and the accuracy of using nuclear 18S rRNA genes for the classification of organisms in the subclass Copepoda. We suggest that sample identification accuracy should be considered when a molecular sequence divergence threshold is used for taxonomic identification, and that the lowest similarity threshold should be determined based on a pre-designated level of acceptable accuracy.

  15. Modified TRIzol Method for RNA and DNA Co-extraction from Blood%改良TRIzol法同步提取血液RNA和DNA

    Institute of Scientific and Technical Information of China (English)

    秦娟娟; 路志勇; 焦章平; 朱晓君; 王颖希; 唐晖


    Objective To establish a new method for RNA and DNA co-extraction from the same sample by TRIzol reagent.Methods After the aqueous phase which contained total RNA was removed by traditional TRIzol method,the values of pH of the interphase phase and organic phase were adjusted.The DNA was precipitated with ethanol and purified with DNA IQTM system.The purified DNA was measured in quality and quantity.As the template,it was amplified and typed by PCR-STR.The data was compared with that extracted by traditional TRIzol method.Results The DNA extracted by this modified method showed a better result of quality and quantity than that by traditional TRIzol method and a good STR typing.Conclusion The modified TRIzol method is advisable and reliable to simultaneously extract both DNA and RNA from the same sample.It could be used for individual identification and paternity testing to satisfy the need of forensic science.%目的 建立一种应用TRIzol试剂针对同一生物检材在提取总RNA的同时又能提取DNA的新方法. 方法 使用传统TRIzol法将含有总RNA的水相移除,调节中间层和有机相的pH值,乙醇沉淀DNA,运用DNA IQTM system试剂盒纯化DNA.检测DNA纯度和质量浓度,并以之为模板进行PCR-STR分型.与传统TRIzol法提取的基因组DNA进行比较. 结果 改良TRIzol法提取的基因组DNA较传统TRIzol法提取的基因组DNA纯度和质量浓度高,扩增后STR分型良好. 结论 改良TRIzol法可靠易行,能够实现同一样本同时提取RNA和DNA的目的,可以满足法庭科学个体识别和亲缘鉴定的要求.

  16. Extraction of MHD Signal Based on Wavelet Transform

    Institute of Scientific and Technical Information of China (English)

    赵晴初; 赵彤; 李旻; 黄胜华; 徐佩霞


    Mirnov signals mixed with interferences are a kind of non-stationary signal. It can not obtain satisfactory effects to extract MHD signals from mirnov signals by Fourier Transform. This paper suggests that the wavelet transform can be used to treat mirnov signals. Theoretical analysis and experimental result have indicated that using the time-frequency analysis characteristics of the wavelet transform to filter mirnov signals can remove effectively interferences and extract useful MHD signals.

  17. Microeukaryotic community and oxygen response in rice field soil revealed using a combined rRNA-gene and rRNA-based approach. (United States)

    Murase, Jun; Takenouchi, Yuriko; Iwasaki, Kazufumi; Kimura, Makoto


    Irrigated rice field soil is subjected to frequent changes in oxygen status due to the water regime by agricultural management. In this study, the community response of microeukaryotes in rice field soil to the oxygen status was explored in a microcosm experiment under defined conditions. Water-saturated soil was incubated under a two-level factorial design of oxygen and organic enrichment with plant residue. The eukaryotic microbial community composition, which was either present or potentially active in the soils, was analyzed using denaturing gradient gel electrophoresis (DGGE) targeting the 18S rRNA gene or reverse-transcribed 18S rRNA. Oxygen availability was a primary factor shaping the microeukaryotic community in both DNA- and RNA-based analyses, revealing a shift within a week of incubation. Plant residue also affected the microeukaryotic community, which was more notable in the active community showing rRNA expression with time. Sequences of amplicons in DGGE bands indicated that protozoa (ciliates, flagellates, and amoebae) were the most prominent microeukaryotes in water-saturated rice field soil both in DNA- and RNA-based analyses. The use of a modified primer for soil protozoa suggested the functional importance of Heterolobosea amoeba in rice field soil, particularly in anoxic soil with organic enrichment.

  18. IsoLasso: A LASSO Regression Approach to RNA-Seq Based Transcriptome Assembly (United States)

    Li, Wei; Feng, Jianxing; Jiang, Tao

    The new second generation sequencing technology revolutionizes many biology related research fields, and posts various computational biology challenges. One of them is transcriptome assembly based on RNA-Seq data, which aims at reconstructing all full-length mRNA transcripts simultaneously from millions of short reads. In this paper, we consider three objectives in transcriptome assembly: the maximization of prediction accuracy, minimization of interpretation, and maximization of completeness. The first objective, the maximization of prediction accuracy, requires that the estimated expression levels based on assembled transcripts should be as close as possible to the observed ones for every expressed region of the genome. The minimization of interpretation follows the parsimony principle to seek as few transcripts in the prediction as possible. The third objective, the maximization of completeness, requires that the maximum number of mapped reads (or "expressed segments" in gene models) be explained by (i.e., contained in) the predicted transcripts in the solution. Based on the above three objectives, we present IsoLasso, a new RNA-Seq based transcriptome assembly tool. IsoLasso is based on the well-known LASSO algorithm, a multivariate regression method designated to seek a balance between the maximization of prediction accuracy and the minimization of interpretation. By including some additional constraints in the quadratic program involved in LASSO, IsoLasso is able to make the set of assembled transcripts as complete as possible. Experiments on simulated and real RNA-Seq datasets show that IsoLasso achieves higher sensitivity and precision simultaneously than the state-of-art transcript assembly tools.

  19. Method for total RNA rapid extraction from mature tissues of fieldgrown apple%田间苹果成熟组织RNA高效提取方法

    Institute of Scientific and Technical Information of China (English)

    焦朝霞; 邵建柱; 孙建设


    The mature leaves of apple trees are rich in polyhexose, polyphenols and other secondary metabolism compounds which prevent RNA extraction. The genomic BNA samples of mature leaves and phloem of field-grown apple in different seasons were isolated by the improved method. Total RNA of high purity, higher content and good integrity could be obtained within about 2 hours by this method and could be well used for RT-PCR. The repetitious experiments indicated that this method was applicable to rapid isolation of RNA from apple trees for simple operation, rapidness, greatly time saving and reduction of using poisonous chemicals. It can be used to detect the viruses from a great amount of samples and can be widely used for research on viruses from fruit trees of various species in different seasons.%针对田间苹果成熟组织富含多酚、多糖和大量次生代谢产物的特点,建立了适合田间苹果不同组织的高效RNA提取方法,以不同季节田间生长的苹果叶片和皮层为材料,用此方法均能在2 h左右完成超过10个样品的总RNA提取,质量高、完整性好的总RNA,可用于田间苹果病毒RT-PCR检测,获得良好效果.经反复大量试验证实,该方法适合田间苹果不同组织的RNA快速提取,操作简便、快捷、高效,且使其不受季节限制,有效地提高了田间果树病毒检测以及其他分子生物学研究的效率.

  20. A grammar based methodology for structural motif finding in ncRNA database search. (United States)

    Quest, Daniel; Tapprich, William; Ali, Hesham


    In recent years, sequence database searching has been conducted through local alignment heuristics, pattern-matching, and comparison of short statistically significant patterns. While these approaches have unlocked many clues as to sequence relationships, they are limited in that they do not provide context-sensitive searching capabilities (e.g. considering pseudoknots, protein binding positions, and complementary base pairs). Stochastic grammars (hidden Markov models HMMs and stochastic context-free grammars SCFG) do allow for flexibility in terms of local context, but the context comes at the cost of increased computational complexity. In this paper we introduce a new grammar based method for searching for RNA motifs that exist within a conserved RNA structure. Our method constrains computational complexity by using a chain of topology elements. Through the use of a case study we present the algorithmic approach and benchmark our approach against traditional methods.

  1. Trends in evolution of 5S rRNA of deuterostomes: bases and homogeneous clusters

    Directory of Open Access Journals (Sweden)

    Sandra Maria Rodrigues Subacius


    Full Text Available Evolution of metazoan 5S rRNA sequences was analyzed through base composition and types, location and frequency of clustered bases. Characters from sequences of protostomes did not show regular trends as compared with paleontology dating or organism complexity. Trends of increasing G and C, stronger in G clusters, and decreasing A and U, were detected in deuterostomes, in parallel with evolution of complexity. The multifunctional domain 71-104 was highlighted among conserved stretches. Clusters of C were typical of helices. Those of G were longer, extending from helices into loops or related to bulges, which is suggestive of functional significance. Deuterostomian trends were installed early in the lineage and reached full development in aquatic organisms, not increasing further after reptiles. It can be suggested that ribosomal RNA structures participated in deuterostomian high regulatory complexity, either specifically or as part of the widespread processes of chromosomal regionalization.

  2. Tetrahedral DNA nanostructure-based microRNA biosensor coupled with catalytic recycling of the analyte. (United States)

    Miao, Peng; Wang, Bidou; Chen, Xifeng; Li, Xiaoxi; Tang, Yuguo


    MicroRNAs are not only important regulators of a wide range of cellular processes but are also identified as promising disease biomarkers. Due to the low contents in serum, microRNAs are always difficult to detect accurately . In this study, an electrochemical biosensor for ultrasensitive detection of microRNA based on tetrahedral DNA nanostructure is developed. Four DNA single strands are engineered to form a tetrahedral nanostructure with a pendant stem-loop and modified on a gold electrode surface, which largely enhances the molecular recognition efficiency. Moreover, taking advantage of strand displacement polymerization, catalytic recycling of microRNA, and silver nanoparticle-based solid-state Ag/AgCl reaction, the proposed biosensor exhibits high sensitivity with the limit of detection down to 0.4 fM. This biosensor shows great clinical value and may have practical utility in early diagnosis and prognosis of certain diseases.

  3. Rank-Based miRNA Signatures for Early Cancer Detection

    Directory of Open Access Journals (Sweden)

    Mario Lauria


    Full Text Available We describe a new signature definition and analysis method to be used as biomarker for early cancer detection. Our new approach is based on the construction of a reference map of transcriptional signatures of both healthy and cancer affected individuals using circulating miRNA from a large number of subjects. Once such a map is available, the diagnosis for a new patient can be performed by observing the relative position on the map of his/her transcriptional signature. To demonstrate its efficacy for this specific application we report the results of the application of our method to published datasets of circulating miRNA, and we quantify its performance compared to current state-of-the-art methods. A number of additional features make this method an ideal candidate for large-scale use, for example, as a mass screening tool for early cancer detection or for at-home diagnostics. Specifically, our method is minimally invasive (because it works well with circulating miRNA, it is robust with respect to lab-to-lab protocol variability and batch effects (it requires that only the relative ranking of expression value of miRNA in a profile be accurate not their absolute values, and it is scalable to a large number of subjects. Finally we discuss the need for HPC capability in a widespread application of our or similar methods.

  4. Viral evasion of a bacterial suicide system by RNA-based molecular mimicry enables infectious altruism. (United States)

    Blower, Tim R; Evans, Terry J; Przybilski, Rita; Fineran, Peter C; Salmond, George P C


    Abortive infection, during which an infected bacterial cell commits altruistic suicide to destroy the replicating bacteriophage and protect the clonal population, can be mediated by toxin-antitoxin systems such as the Type III protein-RNA toxin-antitoxin system, ToxIN. A flagellum-dependent bacteriophage of the Myoviridae, ΦTE, evolved rare mutants that "escaped" ToxIN-mediated abortive infection within Pectobacterium atrosepticum. Wild-type ΦTE encoded a short sequence similar to the repetitive nucleotide sequence of the RNA antitoxin, ToxI, from ToxIN. The ΦTE escape mutants had expanded the number of these "pseudo-ToxI" genetic repeats and, in one case, an escape phage had "hijacked" ToxI from the plasmid-borne toxIN locus, through recombination. Expression of the pseudo-ToxI repeats during ΦTE infection allowed the phage to replicate, unaffected by ToxIN, through RNA-based molecular mimicry. This is the first example of a non-coding RNA encoded by a phage that evolves by selective expansion and recombination to enable viral suppression of a defensive bacterial suicide system. Furthermore, the ΦTE escape phages had evolved enhanced capacity to transduce replicons expressing ToxIN, demonstrating virus-mediated horizontal transfer of genetic altruism.

  5. Viral evasion of a bacterial suicide system by RNA-based molecular mimicry enables infectious altruism.

    Directory of Open Access Journals (Sweden)

    Tim R Blower

    Full Text Available Abortive infection, during which an infected bacterial cell commits altruistic suicide to destroy the replicating bacteriophage and protect the clonal population, can be mediated by toxin-antitoxin systems such as the Type III protein-RNA toxin-antitoxin system, ToxIN. A flagellum-dependent bacteriophage of the Myoviridae, ΦTE, evolved rare mutants that "escaped" ToxIN-mediated abortive infection within Pectobacterium atrosepticum. Wild-type ΦTE encoded a short sequence similar to the repetitive nucleotide sequence of the RNA antitoxin, ToxI, from ToxIN. The ΦTE escape mutants had expanded the number of these "pseudo-ToxI" genetic repeats and, in one case, an escape phage had "hijacked" ToxI from the plasmid-borne toxIN locus, through recombination. Expression of the pseudo-ToxI repeats during ΦTE infection allowed the phage to replicate, unaffected by ToxIN, through RNA-based molecular mimicry. This is the first example of a non-coding RNA encoded by a phage that evolves by selective expansion and recombination to enable viral suppression of a defensive bacterial suicide system. Furthermore, the ΦTE escape phages had evolved enhanced capacity to transduce replicons expressing ToxIN, demonstrating virus-mediated horizontal transfer of genetic altruism.

  6. Fine-grained parallelism accelerating for RNA secondary structure prediction with pseudoknots based on FPGA. (United States)

    Xia, Fei; Jin, Guoqing


    PKNOTS is a most famous benchmark program and has been widely used to predict RNA secondary structure including pseudoknots. It adopts the standard four-dimensional (4D) dynamic programming (DP) method and is the basis of many variants and improved algorithms. Unfortunately, the O(N(6)) computing requirements and complicated data dependency greatly limits the usefulness of PKNOTS package with the explosion in gene database size. In this paper, we present a fine-grained parallel PKNOTS package and prototype system for accelerating RNA folding application based on FPGA chip. We adopted a series of storage optimization strategies to resolve the "Memory Wall" problem. We aggressively exploit parallel computing strategies to improve computational efficiency. We also propose several methods that collectively reduce the storage requirements for FPGA on-chip memory. To the best of our knowledge, our design is the first FPGA implementation for accelerating 4D DP problem for RNA folding application including pseudoknots. The experimental results show a factor of more than 50x average speedup over the PKNOTS-1.08 software running on a PC platform with Intel Core2 Q9400 Quad CPU for input RNA sequences. However, the power consumption of our FPGA accelerator is only about 50% of the general-purpose micro-processors.

  7. Long noncoding RNA expression signature to predict platinum-based chemotherapeutic sensitivity of ovarian cancer patients. (United States)

    Liu, Rong; Zeng, Ying; Zhou, Cheng-Fang; Wang, Ying; Li, Xi; Liu, Zhao-Qian; Chen, Xiao-Ping; Zhang, Wei; Zhou, Hong-Hao


    Dysregulated long noncoding RNAs (lncRNAs) are potential markers of several tumor prognoses. This study aimed to develop a lncRNA expression signature that can predict chemotherapeutic sensitivity for patients with advanced stage and high-grade serous ovarian cancer (HGS-OvCa) treated with platinum-based chemotherapy. The lncRNA expression profiles of 258 HGS-OvCa patients from The Cancer Genome Atlas were analyzed. Results revealed that an eight-lncRNA signature was significantly associated with chemosensitivity in the multivariate logistic regression model, which can accurately predict the chemosensitivity of patients [Area under curve (AUC) = 0.83]. The association of a chemosensitivity predictor with molecular subtypes indicated the excellent prognosis performance of this marker in differentiated, mesenchymal, and immunoreactive subtypes (AUC > 0.8). The significant correlation between ZFAS1 expression and chemosensitivity was confirmed in 233 HGS-OvCa patients from the Gene Expression Omnibus datasets (GSE9891, GSE63885, and GSE51373). In vitro experiments demonstrated that the ZFAS1 expression was upregulated by cisplatin in A2008, HeyA8, and HeyC2 cell lines. This finding suggested that ZFAS1 may participate in platinum resistance. Therefore, the evaluation of the eight-lncRNA signature may be clinically implicated in the selection of platinum-resistant HGS-OvCa patients. The role of ZFAS1 in platinum resistance should be further investigated.

  8. Spliceosomal small nuclear RNAs of Tetrahymena thermophila and some possible snRNA-snRNA base-pairing interactions

    DEFF Research Database (Denmark)

    Orum, H; Nielsen, Henrik; Engberg, J


    We have identified and characterized the full set of spliceosomal small nuclear RNAs (snRNAs; U1, U2, U4, U5 and U6) from the ciliated protozoan Tetrahymena thermophila. With the exception of U4 snRNA, the sizes of the T. thermophila snRNAs are closely similar to their metazoan homologues. The T...

  9. Comparison of different methods for DNA-free RNA isolation from SK-N-MC neuroblastoma

    Directory of Open Access Journals (Sweden)

    Ferreira Ricardo B


    Full Text Available Abstract Background RNA quality and quantity are important factors for ensuring the accuracy of gene expression analysis and other RNA-based downstream applications. Extraction of high quality nucleic acids is difficult from neuronal cells and brain tissues as they are particularly rich in lipids. In addition, most common RNA extraction methods are phenol-based, resulting in RNA that may be incompatible with downstream applications such as gene expression. Findings In this work, a comparative analysis of the RNA quality obtained from SK-N-MC cells was performed using six commonly used RNA isolation kits: two phenol-based kits and four non-phenol based kits. The non-phenol based kits tested AxyPrep Multisource Total RNA Miniprep, RNeasy® Mini, EasySpin and Ilustra RNAspin Mini RNA Isolation, all performed well and resulted in the isolation of high quality RNA, as evaluated by A260/A280. The RNA extracted with AxyPrep Multisource Total RNA Miniprep, RNeasy® Mini and EasySpin provided the highest RNA yields. In particular, the RNA isolated by AxyPrep Multisource Total RNA Miniprep Kit did not show any detectable genomic DNA contamination even without previous DNase treatment or after RNA direct PCR amplification using universal 18S primers. Conclusions The RNA extracted from SK-N-MC cells with AxyPrep Multisource Total RNA Miniprep Kit was superior with respect to the RNA quality and concentration. This kit does not use aggressive organic solvents and RNA free of genomic DNA was isolated without the need for DNase treatment.

  10. PCR-based bioprospecting for homing endonucleases in fungal mitochondrial rRNA genes. (United States)

    Hafez, Mohamed; Guha, Tuhin Kumar; Shen, Chen; Sethuraman, Jyothi; Hausner, Georg


    Fungal mitochondrial genomes act as "reservoirs" for homing endonucleases. These enzymes with their DNA site-specific cleavage activities are attractive tools for genome editing and gene therapy applications. Bioprospecting and characterization of naturally occurring homing endonucleases offers an alternative to synthesizing artificial endonucleases. Here, we describe methods for PCR-based screening of fungal mitochondrial rRNA genes for homing endonuclease encoding sequences, and we also provide protocols for the purification and biochemical characterization of putative native homing endonucleases.

  11. MicroRNA-Based Therapy in Animal Models of Selected Gastrointestinal Cancers



    Gastrointestinal cancer accounts for the 20 most frequent cancer diseases worldwide and there is a constant urge to bring new therapeutics with new mechanism of action into the clinical practice. Quantity of in vitro and in vivo evidences indicate, that exogenous change in pathologically imbalanced microRNAs (miRNAs) is capable of transforming the cancer cell phenotype. This review analyzed preclinical miRNA-based therapy attempts in animal models of gastric, pancreatic, gallbladder, and colo...

  12. Knowledge-Driven Event Extraction in Russian: Corpus-Based Linguistic Resources. (United States)

    Solovyev, Valery; Ivanov, Vladimir


    Automatic event extraction form text is an important step in knowledge acquisition and knowledge base population. Manual work in development of extraction system is indispensable either in corpus annotation or in vocabularies and pattern creation for a knowledge-based system. Recent works have been focused on adaptation of existing system (for extraction from English texts) to new domains. Event extraction in other languages was not studied due to the lack of resources and algorithms necessary for natural language processing. In this paper we define a set of linguistic resources that are necessary in development of a knowledge-based event extraction system in Russian: a vocabulary of subordination models, a vocabulary of event triggers, and a vocabulary of Frame Elements that are basic building blocks for semantic patterns. We propose a set of methods for creation of such vocabularies in Russian and other languages using Google Books NGram Corpus. The methods are evaluated in development of event extraction system for Russian.

  13. A silica gel based method for extracting insect surface hydrocarbons. (United States)

    Choe, Dong-Hwan; Ramírez, Santiago R; Tsutsui, Neil D


    Here, we describe a novel method for the extraction of insect cuticular hydrocarbons using silica gel, herein referred to as "silica-rubbing". This method permits the selective sampling of external hydrocarbons from insect cuticle surfaces for subsequent analysis using gas chromatography-mass spectrometry (GC-MS). The cuticular hydrocarbons are first adsorbed to silica gel particles by rubbing the cuticle of insect specimens with the materials, and then are subsequently eluted using organic solvents. We compared the cuticular hydrocarbon profiles that resulted from extractions using silica-rubbing and solvent-soaking methods in four ant and one bee species: Linepithema humile, Azteca instabilis, Camponotus floridanus, Pogonomyrmex barbatus (Hymenoptera: Formicidae), and Euglossa dilemma (Hymenoptera: Apidae). We also compared the hydrocarbon profiles of Euglossa dilemma obtained via silica-rubbing and solid phase microextraction (SPME). Comparison of hydrocarbon profiles obtained by different extraction methods indicates that silica rubbing selectively extracts the hydrocarbons that are present on the surface of the cuticular wax layer, without extracting hydrocarbons from internal glands and tissues. Due to its surface specificity, efficiency, and low cost, this new method may be useful for studying the biology of insect cuticular hydrocarbons.

  14. Surface Electromyography Feature Extraction Based on Wavelet Transform

    Directory of Open Access Journals (Sweden)

    Farzaneh Akhavan Mahdavi


    Full Text Available Considering the vast variety of EMG signal applications such as rehabilitation of people suffering from some mobility limitations, scientists have done much research on EMG control system. In this regard, feature extraction of EMG signal has been highly valued as a significant technique to extract the desired information of EMG signal and remove unnecessary parts. In this study, Wavelet Transform (WT has been applied as the main technique to extract Surface EMG (SEMG features because WT is consistent with the nature of EMG as a nonstationary signal. Furthermore, two evaluation criteria, namely, RES index (the ratio of a Euclidean distance to a standard deviation and scatter plot are recruited to investigate the efficiency of wavelet feature extraction. The results illustrated an improvement in class separability of hand movements in feature space. Accordingly, it has been shown that only the SEMG features extracted from first and second level of WT decomposition by second order of Daubechies family (db2 yielded the best class separability.

  15. Evolutionary patterns of RNA-based duplication in non-mammalian chordates.

    Directory of Open Access Journals (Sweden)

    Ming Chen

    Full Text Available The role of RNA-based duplication, or retroposition, in the evolution of new gene functions in mammals, plants, and Drosophila has been widely reported. However, little is known about RNA-based duplication in non-mammalian chordates. In this study, we screened ten non-mammalian chordate genomes for retrocopies and investigated their evolutionary patterns. We identified numerous retrocopies in these species. Examination of the age distribution of these retrocopies revealed no burst of young retrocopies in ancient chordate species. Upon comparing these non-mammalian chordate species to the mammalian species, we observed that a larger fraction of the non-mammalian retrocopies was under strong evolutionary constraints than mammalian retrocopies are, as evidenced by signals of purifying selection and expression profiles. For the Western clawed frog, Medaka, and Sea squirt, many retrogenes have evolved gonad and brain expression patterns, similar to what was observed in human. Testing of retrogene movement in the Medaka genome, where the nascent sex chrosomes have been well assembled, did not reveal any significant gene movement. Taken together, our analyses demonstrate that RNA-based duplication generates many functional genes and can make a significant contribution to the evolution of non-mammalian genomes.

  16. Effects of Gingko biloba leaf extract on learning, memory, and hippocampal amyloid precursor protein mRNA expressions in diabetic rats

    Institute of Scientific and Technical Information of China (English)

    Xiaofan Zhang; Bo Liang; Zhifeng Liang; Jun Lin


    BACKGROUND: The mechanisms of brain injury Following diabetes could be related to amyloid precursor protein (APP) mRNA overexpression. Studies have shown that Gingko biloba leaf extract (Egb) is effective in promoting functional recovery of the brain after traumatic injury. Egb is also effective in improving central nervous system plasticity and learning and memory functions of the elderly.OBJECTIVE: To study the effects of Egb on learning and memory, as well as hippocampal APP mRNA expression in the brains of diabetic rats, using Morris water maze behavioral testing and reverse transcription polymerase chain reaction (RT-PCR), respectively.DESIGN: Complete random design, controlled experimental study.SETTING: Department of Pharmacology, Pharmaceutical School, Guangxi Medical University.MATERIALS: A total of 70 male Wistar rats (180-220 g), 8 weeks old and specific pathogen free, were used for this study. GbE (containing 24.8% flavone glycosides and 6.2% diterpene lactone) was purchased from Guilin Sitejia Natural Plants Pharmaceutical Factory (Guangxi Province, Lot NO. 200405). Streptozotocin was purchased from Sigma (USA). Protamine zinc insulin injection was purchased from WANBANG Biochemical Pharmaceutical Co., Ltd. (Xuzhou Jiangsu, China).METHODS: The experiment was performed in the Experimental Center of Guangxi Medical University from March to October 2005. ① Experimental intervention: 70 rats were divided randomly into normal control group, diabetic model group (DM group), diabetic model +10 μg/kg insulin group (DM + Ins group), diabetic model + 100 mg/kg ginkgo leaf extract group (DM + Egb high-dose group), and diabetic model + 50 mg/kg ginkgo leaf extract group (DM + Egb low-dose group); there were 14 rats in each group. Rats with an intraperitoneal (I.p.) injection of citrate buffer solution (pH 4.4) served as the control group. To establish the diabetes model, rats were treated with I.p. Injection of 55 mg/kg streptozotocin. Insulin (10 U/kg) was

  17. Application of ionic liquids based enzyme-assisted extraction of chlorogenic acid from Eucommia ulmoides leaves

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Tingting; Sui, Xiaoyu, E-mail:; Li, Li; Zhang, Jie; Liang, Xin; Li, Wenjing; Zhang, Honglian; Fu, Shuang


    A new approach for ionic liquid based enzyme-assisted extraction (ILEAE) of chlorogenic acid (CGA) from Eucommia ulmoides is presented in which enzyme pretreatment was used in ionic liquids aqueous media to enhance extraction yield. For this purpose, the solubility of CGA and the activity of cellulase were investigated in eight 1-alkyl-3-methylimidazolium ionic liquids. Cellulase in 0.5 M [C6mim]Br aqueous solution was found to provide better performance in extraction. The factors of ILEAE procedures including extraction time, extraction phase pH, extraction temperatures and enzyme concentrations were investigated. Moreover, the novel developed approach offered advantages in term of yield and efficiency compared with other conventional extraction techniques. Scanning electronic microscopy of plant samples indicated that cellulase treated cell wall in ionic liquid solution was subjected to extract, which led to more efficient extraction by reducing mass transfer barrier. The proposed ILEAE method would develope a continuous process for enzyme-assisted extraction including enzyme incubation and solvent extraction process. In this research, we propose a novel view for enzyme-assisted extraction of plant active component, besides concentrating on enzyme facilitated cell wall degradation, focusing on improvement of bad permeability of ionic liquids solutions. - Highlights: • An ionic liquid based enzyme-assisted extraction method of natural product was explored. • ILEAE utilizes enzymatic treatment to improve permeability of ionic liquids solution. • Enzyme incubation and solvent extraction process were ongoing simultaneously. • ILEAE process simplified operating process and suitable for more complete extraction.

  18. 辣椒组织总RNA提取方法研究%Study on RNA Extraction from Hot Pepper Tissues

    Institute of Scientific and Technical Information of China (English)

    李燕凌; 李大志



  19. Comparison of RNA Extraction Method from Tomato Fruit%番茄果实RNA提取方法比较

    Institute of Scientific and Technical Information of China (English)

    马敬; 纪鸿飞; 李培



  20. Ionic liquids based microwave-assisted extraction of lichen compounds with quantitative spectrophotodensitometry analysis. (United States)

    Bonny, Sarah; Paquin, Ludovic; Carrié, Daniel; Boustie, Joël; Tomasi, Sophie


    Ionic liquids based extraction method has been applied to the effective extraction of norstictic acid, a common depsidone isolated from Pertusaria pseudocorallina, a crustose lichen. Five 1-alkyl-3-methylimidazolium ionic liquids (ILs) differing in composition of alkyl chain and anion were investigated for extraction efficiency. The extraction amount of norstictic acid was determined after recovery on HPTLC with a spectrophotodensitometer. The proposed approaches (IL-MAE and IL-heat extraction (IL-HE)) have been evaluated in comparison with usual solvents such as tetrahydrofuran in heat-reflux extraction and microwave-assisted extraction (MAE). The results indicated that both the characteristics of the alkyl chain and anion influenced the extraction of polyphenolic compounds. The sulfate-based ILs [C(1)mim][MSO(4)] and [C(2)mim][ESO(4)] presented the best extraction efficiency of norstictic acid. The reduction of the extraction times between HE and MAE (2 h-5 min) and a non-negligible ratio of norstictic acid in total extract (28%) supports the suitability of the proposed method. This approach was successfully applied to obtain additional compounds from other crustose lichens (Pertusaria amara and Ochrolechia parella).

  1. Application of ionic liquids based enzyme-assisted extraction of chlorogenic acid from Eucommia ulmoides leaves. (United States)

    Liu, Tingting; Sui, Xiaoyu; Li, Li; Zhang, Jie; Liang, Xin; Li, Wenjing; Zhang, Honglian; Fu, Shuang


    A new approach for ionic liquid based enzyme-assisted extraction (ILEAE) of chlorogenic acid (CGA) from Eucommia ulmoides is presented in which enzyme pretreatment was used in ionic liquids aqueous media to enhance extraction yield. For this purpose, the solubility of CGA and the activity of cellulase were investigated in eight 1-alkyl-3-methylimidazolium ionic liquids. Cellulase in 0.5 M [C6mim]Br aqueous solution was found to provide better performance in extraction. The factors of ILEAE procedures including extraction time, extraction phase pH, extraction temperatures and enzyme concentrations were investigated. Moreover, the novel developed approach offered advantages in term of yield and efficiency compared with other conventional extraction techniques. Scanning electronic microscopy of plant samples indicated that cellulase treated cell wall in ionic liquid solution was subjected to extract, which led to more efficient extraction by reducing mass transfer barrier. The proposed ILEAE method would develope a continuous process for enzyme-assisted extraction including enzyme incubation and solvent extraction process. In this research, we propose a novel view for enzyme-assisted extraction of plant active component, besides concentrating on enzyme facilitated cell wall degradation, focusing on improvement of bad permeability of ionic liquids solutions.

  2. Social Network Extraction and Analysis Based on Multimodal Dyadic Interaction

    Directory of Open Access Journals (Sweden)

    Bogdan Raducanu


    Full Text Available Social interactions are a very important component in people’s lives. Social network analysis has become a common technique used to model and quantify the properties of social interactions. In this paper, we propose an integrated framework to explore the characteristics of a social network extracted from multimodal dyadic interactions. For our study, we used a set of videos belonging to New York Times’ Blogging Heads opinion blog. The Social Network is represented as an oriented graph, whose directed links are determined by the Influence Model. The links’ weights are a measure of the “influence” a person has over the other. The states of the Influence Model encode automatically extracted audio/visual features from our videos using state-of-the art algorithms. Our results are reported in terms of accuracy of audio/visual data fusion for speaker segmentation and centrality measures used to characterize the extracted social network.

  3. Ontology-based Knowledge Extraction from Hidden Web

    Institute of Scientific and Technical Information of China (English)

    SONG Hui; MA Fan-yuan; LIU Xiao-qiang


    Hidden Web provides great amount of domain-specific data for constructing knowledge services. Most previous knowledge extraction researches ignore the valuable data hidden in Web database, and related works do not refer how to make extracted information available for knowledge system. This paper describes a novel approach to build a domain-specific knowledge service with the data retrieved from Hidden Web. Ontology serves to model the domain knowledge. Queries forms of different Web sites are translated into machine-understandable format, defined knowledge concepts, so that they can be accessed automatically. Also knowledge data are extracted from Web pages and organized in ontology format knowledge. The experiment proves the algorithm achieves high accuracy and the system facilitates constructing knowledge services greatly.

  4. A Karnaugh-Map based fingerprint minutiae extraction method

    Directory of Open Access Journals (Sweden)

    Sunil Kumar Singla


    Full Text Available Fingerprint is one of the most promising method among all the biometric techniques and has been used for thepersonal authentication for a long time because of its wide acceptance and reliability. Features (Minutiae are extracted fromthe fingerprint in question and are compared with the features already stored in the database for authentication. Crossingnumber (CN is the most commonly used minutiae extraction method for fingerprints. In this paper, a new Karnaugh-Mapbased fingerprint minutiae extraction method has been proposed and discussed. In the proposed algorithm the 8 neighborsof a pixel in a 33 window are arranged as 8 bits of a byte and corresponding hexadecimal (hex value is calculated. Thesehex values are simplified using standard Karnaugh-Map (K-map technique to obtain the minimized logical expression.Experiments conducted on the FVC2002/Db1_a database reveals that the developed method is better than the crossingnumber (CN method.

  5. Engineering Structurally Interacting RNA (sxRNA) (United States)

    Doyle, Francis; Lapsia, Sameer; Spadaro, Salvatore; Wurz, Zachary E.; Bhaduri-McIntosh, Sumita; Tenenbaum, Scott A.


    RNA-based three-way junctions (3WJs) are naturally occurring structures found in many functional RNA molecules including rRNA, tRNA, snRNA and ribozymes. 3WJs are typically characterized as resulting from an RNA molecule folding back on itself in cis but could also form in trans when one RNA, for instance a microRNA binds to a second structured RNA, such as a mRNA. Trans-3WJs can influence the final shape of one or both of the RNA molecules and can thus provide a means for modulating the availability of regulatory motifs including potential protein or microRNA binding sites. Regulatory 3WJs generated in trans represent a newly identified regulatory category that we call structurally interacting RNA or sxRNA for convenience. Here we show that they can be rationally designed using familiar cis-3WJ examples as a guide. We demonstrate that an sxRNA “bait” sequence can be designed to interact with a specific microRNA “trigger” sequence, creating a regulatable RNA-binding protein motif that retains its functional activity. Further, we show that when placed downstream of a coding sequence, sxRNA can be used to switch “ON” translation of that sequence in the presence of the trigger microRNA and the amount of translation corresponded with the amount of microRNA present. PMID:28350000

  6. miRNA Isolation from FFPET Specimen: A Technical Comparison of miRNA and Total RNA Isolation Methods. (United States)

    Nagy, Zsófia Brigitta; Wichmann, Barnabás; Kalmár, Alexandra; Barták, Barbara Kinga; Tulassay, Zsolt; Molnár, Béla


    MiRNA remain stable for detection and PCR-based amplification in FFPE tissue samples. Several miRNA extraction kits are available, however miRNA fraction, as part of total RNA can be isolated using total RNA purification methods, as well. Our primary aim was to compare four different miRNA and total RNA isolation methods from FFPE tissues. Further purposes were to evaluate quantitatively and qualitatively the yield of the isolated miRNA. MiRNAs were isolated from normal colorectal cancer FFPE specimens from the same patients. Two miRNA isolation kits (High Pure miRNA Isolation Kit, miRCURY™ RNA Isolation Kit) and two total RNA isolation kits were compared (High Pure RNA Paraffin Kit, MagNA Pure 96 Cellular RNA LV Kit). Quantity and quality were determined, expression analysis was performed by real-time PCR using qPCR Human Panel I + II (Exiqon) method detecting 742 human miRNAs in parallel. The yield of total RNA was found to be higher than miRNA purification protocols (in CRC: Ex: 0203 ± 0021 μg; HPm: 1,45 ± 0,8 μg; HPp: 21,36 ± 4,98 μg; MP: 8,6 ± 5,1 μg). MiRNAs were detected in lower relative quantity of total RNA compared to the miRNA kits. Higher number of miRNAs could be detected by the miRNA isolation kits in comparison to the total RNA isolation methods. (Ex: 497 ± 16; HPm: 542 ± 11; HPp: 332 ± 36; MP: 295 ± 74). Colon specific miRNAs (miR-21-5p;-34-5p) give satisfying results by miRNA isolation kits. Although miRNA can be detected also after total RNA isolation methods, for reliable and reproducible miRNA expression profiling the use of miRNA isolation kits are more suitable.

  7. Distinct summer and winter bacterial communities in the active layer of Svalbard permafrost revealed by DNA- and RNA-based analyses

    Energy Technology Data Exchange (ETDEWEB)

    Schostag, Morten; Stibal, Marek; Jacobsen, Carsten S.; Baelum, Jacob; Tas, Neslihan; Elberling, Bo; Jansson, Janet K.; Semenchuk, Phillip; Prieme, Anders


    The active layer of soil overlaying permafrost in the Arctic is subjected to dramatic annual changes in temperature and soil chemistry, which likely affect bacterial activity and community structure. We studied seasonal variations in the bacterial community of active layer soil from Svalbard (78°N) by co-extracting DNA and RNA from 12 soil cores collected monthly over a year. PCR amplicons of 16S rRNA genes (DNA) and reverse transcribed transcripts (cDNA) were quantified and sequenced to test for the effect of low winter temperature and seasonal variation in concentration of easily degradable organic matter on the bacterial communities. The copy number of 16S rRNA genes and transcripts revealed no distinct seasonal changes indicating potential bacterial activity during winter despite soil temperatures well below -10°C. Multivariate statistical analysis of the bacterial diversity data (DNA and cDNA libraries) revealed a season-based clustering of the samples, and, e.g., the relative abundance of potentially active Cyanobacteria peaked in June and Alphaproteobacteria increased over the summer and then declined from October to November. The structure of the bulk (DNA-based) community was significantly correlated with pH and dissolved organic carbon, while the potentially active (RNA-based) community structure was not significantly correlated with any of the measured soil parameters. A large fraction of the 16S rRNA transcripts was assigned to nitrogen-fixing bacteria (up to 24% in June) and phototrophic organisms (up to 48% in June) illustrating the potential importance of nitrogen fixation in otherwise nitrogen poor Arctic ecosystems and of phototrophic bacterial activity on the soil surface.

  8. Partial base-methylation and other structural differences in the 17 S ribosomal RNA of sycamore cells during growth in cell culture. (United States)

    Miassod, R; Cecchini, J P


    Sycamore (Acer pseudoplatanus L.) cytoplasmic rRNA was investigated in rapidly dividing cells, cells starting mitosis after the lag phase of growth (4 days) induced by deconditioning of the culture medium and also in growth-arrested cells from 10 day-old cultures deprived of exogenous auxin (i.e. exponential, early exponential and 2,4-dichlorophenoxyacetic acid (2,4-D)-deprived cultures). rRNA was extracted and purified from mixed 14C-labelled exponential cultures and 3H-labelled early exponential cultures. A 14C-labelled exponential culture and a 3H-labelled 2,4-D-deprived culture were analyzed in the same way. The 17 S rRNA molecules from both early exponential and 2,4-D-deprived cultures displayed a lower electrophoretic mobility on polyacrylamide gels than those from exponential cultures. Alkaline and acid hydrolysates of purified 17 S rRNA labelled on the phosphate groups or the methyl groups were analyzed on ion-exchange resins. There was no change in the extent of ribose methylation of the molecule from the three different cultures. However, the base methylation of the 17 S rRNA was decreased in early exponential cultures and in 2,4-D-deprived cultures. Part of the molecules synthesized in early exponential cultures specifically lacked 7-methylguanine, N6-methyladenine and N6,N6-dimethyladenine. The possible significance of these changes in the 17 S rRNA were discussed.

  9. Background Knowledge in Learning-Based Relation Extraction (United States)

    Do, Quang Xuan


    In this thesis, we study the importance of background knowledge in relation extraction systems. We not only demonstrate the benefits of leveraging background knowledge to improve the systems' performance but also propose a principled framework that allows one to effectively incorporate knowledge into statistical machine learning models for…

  10. Coastline Extraction from Aerial Images Based on Edge Detection (United States)

    Paravolidakis, V.; Moirogiorgou, K.; Ragia, L.; Zervakis, M.; Synolakis, C.


    Nowadays coastline extraction and tracking of its changes become of high importance because of the climate change, global warming and rapid growth of human population. Coastal areas play a significant role for the economy of the entire region. In this paper we propose a new methodology for automatic extraction of the coastline using aerial images. A combination of a four step algorithm is used to extract the coastline in a robust and generalizable way. First, noise distortion is reduced in order to ameliorate the input data for the next processing steps. Then, the image is segmented into two regions, land and sea, through the application of a local threshold to create the binary image. The result is further processed by morphological operators with the aim that small objects are being eliminated and only the objects of interest are preserved. Finally, we perform edge detection and active contours fitting in order to extract and model the coastline. These algorithmic steps are illustrated through examples, which demonstrate the efficacy of the proposed methodology.

  11. Apect-Based Opinion Extraction From Customer Reviews

    Directory of Open Access Journals (Sweden)

    Amani K Samha


    Full Text Available Text is the main method of communicating informatio n in the digital age. Messages, blogs, news articles, reviews, and opinionated information abounds on the Internet. People commonly purchase products online and post their opinions ab out purchased items. This feedback is displayed publicly to assist others with their purc hasing decisions, creating the need for a mechanism with which to extract and summarize usefu l information for enhancing the decision- making process. Our contribution is to improve the accuracy of extraction by combining different techniques from three major areas, namedD ata Mining, Natural Language Processing techniques and Ontologies. The proposed framework sequentially mines product’s aspects and users’ opinions, groups representative aspects by s imilarity, and generates an output summary. This paper focuses on the task of extracting produc t aspects and users’ opinions by extracting all possible aspects and opinions from reviews usin g natural language, ontology, and frequent “tag”sets. The proposed framework, when compared w ith an existing baseline model, yielded promising results.

  12. Rule set transferability for object-based feature extraction

    NARCIS (Netherlands)

    Anders, N.S.; Seijmonsbergen, Arie C.; Bouten, Willem


    Cirques are complex landforms resulting from glacial erosion and can be used to estimate Equilibrium Line Altitudes and infer climate history. Automated extraction of cirques may help research on glacial geomorphology and climate change. Our objective was to test the transferability of an object-

  13. microRNA-based diagnostics and therapy in cardiovascular disease-Summing up the facts. (United States)

    Schulte, Christian; Zeller, Tanja


    Circulating microRNAs (miRNAs) are discussed as potential disease-specific biomarkers in cardiovascular disease. Their diagnostic value has been examined in numerous studies and animal models with respect to coronary artery disease (CAD) and myocardial infarction (MI) and the prognostic abilities of circulating miRNAs in risk stratification of future disease have been evaluated. Various miRNAs are described to complement protein-based biomarkers or classical risk factors in the diagnosis of CAD or MI and even represent potential new biomarkers in the discrimination of unstable angina pectoris (UAP). Signatures consisting of sets of multiple miRNAs seem to improve the predictive power compared to single miRNAs. Furthermore, the emerging field of miRNA-based therapeutics has reached cardiovascular research. The first promising in vitro results are raising hope for future clinical application. However, methods and material used for RNA isolation, miRNA detection and normalization steps still lack ways of standardization and need to be considered carefully. This article reviews the current knowledge of miRNAs in cardiovascular disease focusing on CAD and MI and will provide an overview regarding the use of circulating miRNAs as biomarkers and potential therapeutic targets in the field of CAD.

  14. MicroRNA-based Therapy in Animal Models of Selected Gastrointestinal Cancers

    Directory of Open Access Journals (Sweden)

    Jana Merhautova


    Full Text Available Gastrointestinal cancer accounts for the 20 most frequent cancer diseases worldwide and there is a constant urge to bring new therapeutics with new mechanism of action into the clinical practice. Quantity of in vitro and in vivo evidences indicate, that exogenous change in pathologically imbalanced microRNAs (miRNAs is capable of transforming the cancer cell phenotype. This review analyzed preclinical miRNA-based therapy attempts in animal models of gastric, pancreatic, gallbladder, and colorectal cancer. From more than 400 original articles, 26 was found to assess the effect of miRNA mimics, precursors, expression vectors, or inhibitors administered locally or systemically being an approach with relatively high translational potential. We have focused on mapping available information on animal model used (animal strain, cell line, xenograft method, pharmacological aspects (oligonucleotide chemistry, delivery system, posology, route of administration and toxicology assessments. We also summarize findings in the field pharmacokinetics and toxicity of miRNA-based therapy.□

  15. Messenger RNA- versus retrovirus-based induced pluripotent stem cell reprogramming strategies: analysis of genomic integrity. (United States)

    Steichen, Clara; Luce, Eléanor; Maluenda, Jérôme; Tosca, Lucie; Moreno-Gimeno, Inmaculada; Desterke, Christophe; Dianat, Noushin; Goulinet-Mainot, Sylvie; Awan-Toor, Sarah; Burks, Deborah; Marie, Joëlle; Weber, Anne; Tachdjian, Gérard; Melki, Judith; Dubart-Kupperschmitt, Anne


    The use of synthetic messenger RNAs to generate human induced pluripotent stem cells (iPSCs) is particularly appealing for potential regenerative medicine applications, because it overcomes the common drawbacks of DNA-based or virus-based reprogramming strategies, including transgene integration in particular. We compared the genomic integrity of mRNA-derived iPSCs with that of retrovirus-derived iPSCs generated in strictly comparable conditions, by single-nucleotide polymorphism (SNP) and copy number variation (CNV) analyses. We showed that mRNA-derived iPSCs do not differ significantly from the parental fibroblasts in SNP analysis, whereas retrovirus-derived iPSCs do. We found that the number of CNVs seemed independent of the reprogramming method, instead appearing to be clone-dependent. Furthermore, differentiation studies indicated that mRNA-derived iPSCs differentiated efficiently into hepatoblasts and that these cells did not load additional CNVs during differentiation. The integration-free hepatoblasts that were generated constitute a new tool for the study of diseased hepatocytes derived from patients' iPSCs and their use in the context of stem cell-derived hepatocyte transplantation. Our findings also highlight the need to conduct careful studies on genome integrity for the selection of iPSC lines before using them for further applications.

  16. Monitoring of the microbiota of fermented sausages by culture independent rRNA-based approaches. (United States)

    Greppi, Anna; Ferrocino, Ilario; La Storia, Antonietta; Rantsiou, Kalliopi; Ercolini, Danilo; Cocolin, Luca


    In Italy, fermented sausages (called "salami") are consumed in large quantities. Salami samples from a local meat factory in the area of Torino were analyzed at 0, 3, 7, 30 and 45 days of ripening. Swab samples from the production environment were also collected at the beginning of the experiment. The diversity of metabolically active microbiota occurring during the natural fermentation of salami was evaluated by using RT-PCR-DGGE coupled with RNA-based pyrosequencing of the 16S rRNA gene. A culture-dependent approach was also applied to identify and characterize isolated Staphylococcaceae and LAB populations. Staphylococcus succinus, Staphylococcus xylosus and Lactobacillus sakei were the species most frequently isolated during the maturation time. Rep-PCR analysis showed that S. succinus and S. xylosus isolated from swabs and salami samples clustered together, suggesting possible contamination during the production process. RT-PCR-DGGE and rRNA-based pyrosequencing showed that the metabolically active populations were dominated by S. succinus, Lb. sakei and Leuconostoc carnosum. In this specific case study, only a few species belonging to Staphylococcaceae, Lactobacillaceae and Leuconostocaceae may be metabolically active and contribute to determine the final characteristics of the products.

  17. Double-stranded RNA transcribed from vector-based oligodeoxynucleotide acts as transcription factor decoy

    Energy Technology Data Exchange (ETDEWEB)

    Xiao, Xiao [State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province (China); Gang, Yi [State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province (China); Department of Infectious Diseases, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038, Shaanxi Province (China); Wang, Honghong [No. 518 Hospital of Chinese People’s Liberation Army, Xi’an 710043, Shaanxi Province (China); Wang, Jiayin [The Genome Institute, Washington University in St. Louis, St. Louis, MO 63108 (United States); Zhao, Lina [Department of Radiation Oncology, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province (China); Xu, Li, E-mail: [State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province (China); Liu, Zhiguo, E-mail: [State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province (China)


    Highlights: • A shRNA vector based transcription factor decoy, VB-ODN, was designed. • VB-ODN for NF-κB inhibited cell viability in HEK293 cells. • VB-ODN inhibited expression of downstream genes of target transcription factors. • VB-ODN may enhance nuclear entry ratio for its feasibility of virus production. - Abstract: In this study, we designed a short hairpin RNA vector-based oligodeoxynucleotide (VB-ODN) carrying transcription factor (TF) consensus sequence which could function as a decoy to block TF activity. Specifically, VB-ODN for Nuclear factor-κB (NF-κB) could inhibit cell viability and decrease downstream gene expression in HEK293 cells without affecting expression of NF-κB itself. The specific binding between VB-ODN produced double-stranded RNA and NF-κB was evidenced by electrophoretic mobility shift assay. Moreover, similar VB-ODNs designed for three other TFs also inhibit their downstream gene expression but not that of themselves. Our study provides a new design of decoy for blocking TF activity.

  18. A novel technique for extracting clouds base height using ground based imaging

    Directory of Open Access Journals (Sweden)

    E. Hirsch


    Full Text Available The height of a cloud in the atmospheric column is a key parameter in its characterization. Several remote sensing techniques (passive and active, either ground-based or on space-borne platforms and in-situ measurements are routinely used in order to estimate top and base heights of clouds. In this article we present a novel method that combines thermal imaging from the ground and sounded wind profile in order to derive the cloud base height. This method is independent of cloud types, making it efficient for both low boundary layer and high clouds. In addition, using thermal imaging ensures extraction of clouds' features during daytime as well as at nighttime. The proposed technique was validated by comparison to active sounding by ceilometers (which is a standard ground based method, to lifted condensation level (LCL calculations, and to MODIS products obtained from space. As all passive remote sensing techniques, the proposed method extracts only the height of the lowest cloud layer, thus upper cloud layers are not detected. Nevertheless, the information derived from this method can be complementary to space-borne cloud top measurements when deep-convective clouds are present. Unlike techniques such as LCL, this method is not limited to boundary layer clouds, and can extract the cloud base height at any level, as long as sufficient thermal contrast exists between the radiative temperatures of the cloud and its surrounding air parcel. Another advantage of the proposed method is its simplicity and modest power needs, making it particularly suitable for field measurements and deployment at remote locations. Our method can be further simplified for use with visible CCD or CMOS camera (although nighttime clouds will not be observed.

  19. An RNA secondary structure prediction method based on minimum and suboptimal free energy structures. (United States)

    Fu, Haoyue; Yang, Lianping; Zhang, Xiangde


    The function of an RNA-molecule is mainly determined by its tertiary structures. And its secondary structure is an important determinant of its tertiary structure. The comparative methods usually give better results than the single-sequence methods. Based on minimum and suboptimal free energy structures, the paper presents a novel method for predicting conserved secondary structure of a group of related RNAs. In the method, the information from the known RNA structures is used as training data in a SVM (Support Vector Machine) classifier. Our method has been tested on the benchmark dataset given by Puton et al. The results show that the average sensitivity of our method is higher than that of other comparative methods such as CentroidAlifold, MXScrana, RNAalifold, and TurboFold.

  20. Ratiometric FRET-based detection of DNA and micro-RNA in solution

    Energy Technology Data Exchange (ETDEWEB)

    Matveeva, Evgenia G., E-mail: ematveev@hsc.unt.ed [Center for Commercialization of Fluorescence Technologies, University of North Texas Health Science Center, Department of Molecular Biology and Immunology and Department of Cell Biology and Genetics, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107 (United States); Gryczynski, Zygmunt [Center for Commercialization of Fluorescence Technologies, University of North Texas Health Science Center, Department of Molecular Biology and Immunology and Department of Cell Biology and Genetics, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107 (United States); Stewart, Donald R. [Omm Scientific, Inc., 2600 N. Stemmons Freeway, Suite 129, Dallas, TX 75207 (United States); Gryczynski, Ignacy [Center for Commercialization of Fluorescence Technologies, University of North Texas Health Science Center, Department of Molecular Biology and Immunology and Department of Cell Biology and Genetics, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107 (United States)


    A ratiometric method for detecting DNA oligomers in bulk solution based on Foerster resonance energy transfer (FRET) is described. The two fluorescence signals (green and red), originating from Cy3 (donor, green) and Cy5 (acceptor, red) labels, are simultaneously detected from the pre-hybridized Cy3oligomerY:Cy5oligomerX system. The ratio of red to green intensities is sensitive to the presence of the single-stranded complimentary oligomer, which replaces single-stranded Cy3oligomerY in the donor:acceptor complex and perturbs the FRET. The detection scheme is generally applicable to the detection of DNA and RNA, and particularly micro-RNA. The proposed method is applicable to various double-stranded various lengths targets (manipulation of the sample preparation conditions, such as temperature, incubation time, denaturizing agent, may be needed).

  1. Feature Extraction with Ordered Mean Values for Content Based Image Classification

    Directory of Open Access Journals (Sweden)

    Sudeep Thepade


    Full Text Available Categorization of images into meaningful classes by efficient extraction of feature vectors from image datasets has been dependent on feature selection techniques. Traditionally, feature vector extraction has been carried out using different methods of image binarization done with selection of global, local, or mean threshold. This paper has proposed a novel technique for feature extraction based on ordered mean values. The proposed technique was combined with feature extraction using discrete sine transform (DST for better classification results using multitechnique fusion. The novel methodology was compared to the traditional techniques used for feature extraction for content based image classification. Three benchmark datasets, namely, Wang dataset, Oliva and Torralba (OT-Scene dataset, and Caltech dataset, were used for evaluation purpose. Performance measure after evaluation has evidently revealed the superiority of the proposed fusion technique with ordered mean values and discrete sine transform over the popular approaches of single view feature extraction methodologies for classification.

  2. Analysis of energy-based algorithms for RNA secondary structure prediction

    Directory of Open Access Journals (Sweden)

    Hajiaghayi Monir


    Full Text Available Abstract Background RNA molecules play critical roles in the cells of organisms, including roles in gene regulation, catalysis, and synthesis of proteins. Since RNA function depends in large part on its folded structures, much effort has been invested in developing accurate methods for prediction of RNA secondary structure from the base sequence. Minimum free energy (MFE predictions are widely used, based on nearest neighbor thermodynamic parameters of Mathews, Turner et al. or those of Andronescu et al. Some recently proposed alternatives that leverage partition function calculations find the structure with maximum expected accuracy (MEA or pseudo-expected accuracy (pseudo-MEA methods. Advances in prediction methods are typically benchmarked using sensitivity, positive predictive value and their harmonic mean, namely F-measure, on datasets of known reference structures. Since such benchmarks document progress in improving accuracy of computational prediction methods, it is important to understand how measures of accuracy vary as a function of the reference datasets and whether advances in algorithms or thermodynamic parameters yield statistically significant improvements. Our work advances such understanding for the MFE and (pseudo-MEA-based methods, with respect to the latest datasets and energy parameters. Results We present three main findings. First, using the bootstrap percentile method, we show that the average F-measure accuracy of the MFE and (pseudo-MEA-based algorithms, as measured on our largest datasets with over 2000 RNAs from diverse families, is a reliable estimate (within a 2% range with high confidence of the accuracy of a population of RNA molecules represented by this set. However, average accuracy on smaller classes of RNAs such as a class of 89 Group I introns used previously in benchmarking algorithm accuracy is not reliable enough to draw meaningful conclusions about the relative merits of the MFE and MEA-based algorithms

  3. Growth and nitrogen metabolism of sea bass fed graded levels of nucleic acid nitrogen from yeast or RNA extract as partial substitute for protein nitrogen from fish meal

    Directory of Open Access Journals (Sweden)

    S. Kaushik


    Full Text Available Some studies carried out in mammalian models have shown de novo synthesis and salvage of nucleotides to be a costly metabolic process and a dietary supplementation with nucleic acids (NA or nucleotides has been suggested to result in a protein sparing action (Sanderson and He, 1994. On the other hand, high levels of dietary NA could have toxic effects and lead to disturbance in protein, lipid and carbohydrate metabolism in monogastric animals lacking uricase activity, an enzyme involved in NA degradation (Clifford and Story, 1976. So far, there is no clear indication of such effects in fish fed nucleic acid-enriched diets (Tacon and Cooke, 1980; Rumsey et al., 1992; Fournier et al., 2002. The aim of this experiment was to investigate growth response and N metabolism in juvenile sea bass (D. labrax fed diets supplying graded levels of nucleic acid N from dry brewer's yeast or RNA extract as partial substitutes for protein nitrogen provided by fish meal.

  4. Factors affecting reproducibility between genome-scale siRNA-based screens (United States)

    Barrows, Nicholas J.; Le Sommer, Caroline; Garcia-Blanco, Mariano A.; Pearson, James L.


    RNA interference-based screening is a powerful new genomic technology which addresses gene function en masse. To evaluate factors influencing hit list composition and reproducibility, we performed two identically designed small interfering RNA (siRNA)-based, whole genome screens for host factors supporting yellow fever virus infection. These screens represent two separate experiments completed five months apart and allow the direct assessment of the reproducibility of a given siRNA technology when performed in the same environment. Candidate hit lists generated by sum rank, median absolute deviation, z-score, and strictly standardized mean difference were compared within and between whole genome screens. Application of these analysis methodologies within a single screening dataset using a fixed threshold equivalent to a p-value ≤ 0.001 resulted in hit lists ranging from 82 to 1,140 members and highlighted the tremendous impact analysis methodology has on hit list composition. Intra- and inter-screen reproducibility was significantly influenced by the analysis methodology and ranged from 32% to 99%. This study also highlighted the power of testing at least two independent siRNAs for each gene product in primary screens. To facilitate validation we conclude by suggesting methods to reduce false discovery at the primary screening stage. In this study we present the first comprehensive comparison of multiple analysis strategies, and demonstrate the impact of the analysis methodology on the composition of the “hit list”. Therefore, we propose that the entire dataset derived from functional genome-scale screens, especially if publicly funded, should be made available as is done with data derived from gene expression and genome-wide association studies. PMID:20625183

  5. Molecular epidemiology of Plasmodium species prevalent in Yemen based on 18 s rRNA

    Directory of Open Access Journals (Sweden)

    A Azazy Ahmed


    Full Text Available Abstract Background Malaria is an endemic disease in Yemen and is responsible for 4.9 deaths per 100,000 population per year and 43,000 disability adjusted life years lost. Although malaria in Yemen is caused mainly by Plasmodium falciparum and Plasmodium vivax, there are no sequence data available on the two species. This study was conducted to investigate the distribution of the Plasmodium species based on the molecular detection and to study the molecular phylogeny of these parasites. Methods Blood samples from 511 febrile patients were collected and a partial region of the 18 s ribosomal RNA (18 s rRNA gene was amplified using nested PCR. From the 86 positive blood samples, 13 Plasmodium falciparum and 4 Plasmodium vivax were selected and underwent cloning and, subsequently, sequencing and the sequences were subjected to phylogenetic analysis using the neighbor-joining and maximum parsimony methods. Results Malaria was detected by PCR in 86 samples (16.8%. The majority of the single infections were caused by P. falciparum (80.3%, followed by P. vivax (5.8%. Mixed infection rates of P. falciparum + P. vivax and P. falciparum + P. malariae were 11.6% and 2.3%, respectively. All P. falciparum isolates were grouped with the strain 3D7, while P. vivax isolates were grouped with the strain Salvador1. Phylogenetic trees based on 18 s rRNA placed the P. falciparum isolates into three sub-clusters and P. vivax into one cluster. Sequence alignment analysis showed 5-14.8% SNP in the partial sequences of the 18 s rRNA of P. falciparum. Conclusions Although P. falciparum is predominant, P. vivax, P. malariae and mixed infections are more prevalent than has been revealed by microscopy. This overlooked distribution should be considered by malaria control strategy makers. The genetic polymorphisms warrant further investigation.

  6. Application of ionic liquid-based microwave-assisted extraction of flavonoids from Scutellaria baicalensis Georgi. (United States)

    Zhang, Qin; Zhao, San-Hu; Chen, Jue; Zhang, Li-Wei


    In the present work, a rapid ionic liquid-based microwave-assisted extraction (ILMAE) method was successfully applied to simultaneous extraction of baicalin, wogonoside, baicalein and wogonin from Scutellaria baicalensis Georgi. A series of 1-alkyl-3-methylirnidazolium ionic liquids with different anions and cations were assessed for extraction efficiency, and 1-octyl-3-methylimidazolium bromide was selected as the optimal solvent. In addition, the parameters of ILMAE procedure for the four flavonoids were optimized, and the optimal ILMAE method was validated in the linearity, stability, precision and recovery. Meanwhile, the microstructures of S. baicalensis powders were observed before and after extraction with the help of a scanning electron microscope (SEM) in order to explore the extraction mechanism, and the activity of the crude enzyme solution from S. baicalensis was determined through the hydrolysis of baicalin. Finally, the extraction yields and extraction time of WaterHRE, WaterMAE, ILHRE and Chp were 5.18% (30min), 8.77% (90s), 16.94% (30min) and 18.58% (3h), respectively. The results indicated that compared with the conventional extraction approaches, ILMAE possessed great advantages in extracting flavonoids, such as the highest extraction yield (22.28%), the shortest extraction time (90s), etc.

  7. Ionic Liquid-Based Microwave-Assisted Extraction of Flavonoids from Bauhinia championii (Benth. Benth.

    Directory of Open Access Journals (Sweden)

    Wei Xu


    Full Text Available An ionic liquids (IL-based microwave-assisted approach for extraction and determination of flavonoids from Bauhinia championii (Benth. Benth. was proposed for the first time. Several ILs with different cations and anions and the microwave-assisted extraction (MAE conditions, including sample particle size, extraction time and liquid-solid ratio, were investigated. Two M 1-butyl-3-methylimidazolium bromide ([bmim] Br solution with 0.80 M HCl was selected as the optimal solvent. Meanwhile the optimized conditions a ratio of liquid to material of 30:1, and the extraction for 10 min at 70 °C. Compared with conventional heat-reflux extraction (CHRE and the regular MAE, IL-MAE exhibited a higher extraction yield and shorter extraction time (from 1.5 h to 10 min. The optimized extraction samples were analysed by LC-MS/MS. IL extracts of Bauhinia championii (Benth.Benth consisted mainly of flavonoids, among which myricetin, quercetin and kaempferol, β-sitosterol, triacontane and hexacontane were identified. The study indicated that IL-MAE was an efficient and rapid method with simple sample preparation. LC-MS/MS was also used to determine the chemical composition of the ethyl acetate/MAE extract of Bauhinia championii (Benth. Benth, and it maybe become a rapid method to determine the composition of new plant extracts.

  8. Discovery of Potent Non-Nucleoside Inhibitors of Dengue Viral RNA-Dependent RNA Polymerase from a Fragment Hit Using Structure-Based Drug Design. (United States)

    Yokokawa, Fumiaki; Nilar, Shahul; Noble, Christian G; Lim, Siew Pheng; Rao, Ranga; Tania, Stefani; Wang, Gang; Lee, Gladys; Hunziker, Jürg; Karuna, Ratna; Manjunatha, Ujjini; Shi, Pei-Yong; Smith, Paul W


    The discovery and optimization of non-nucleoside dengue viral RNA-dependent-RNA polymerase (RdRp) inhibitors are described. An X-ray-based fragment screen of Novartis' fragment collection resulted in the identification of a biphenyl acetic acid fragment 3, which bound in the palm subdomain of RdRp. Subsequent optimization of the fragment hit 3, relying on structure-based design, resulted in a >1000-fold improvement in potency in vitro and acquired antidengue activity against all four serotypes with low micromolar EC50 in cell-based assays. The lead candidate 27 interacts with a novel binding pocket in the palm subdomain of the RdRp and exerts a promising activity against all clinically relevant dengue serotypes.


    Directory of Open Access Journals (Sweden)

    Necla ÖZKAYA


    Full Text Available Automatic fingerprint recognition systems are utilised for personal identification with the use of comparisons of local ridge characteristics and their relationships. Critical stages in personal identification are to extract features automatically, fast and reliably from the input fingerprint images. In this study, a new approach based on artificial neural networks to extract minutiae from fingerprint images is developed and introduced. The results have shown that artificial neural networks achieve the minutiae extraction from fingerprint images with high accuracy.

  10. Heterogeneous Web Data Extraction Algorithm Based On Modified Hidden Conditional Random Fields


    Cui Cheng


    As it is of great importance to extract useful information from heterogeneous Web data, in this paper, we propose a novel heterogeneous Web data extraction algorithm using a modified hidden conditional random fields model. Considering the traditional linear chain based conditional random fields can not effectively solve the problem of complex and heterogeneous Web data extraction, we modify the standard hidden conditional random fields in three aspects, which are 1) Using the hidden Markov mo...

  11. A strategy for developing a hammerhead ribozyme for selective RNA cleavage depending on substitutional RNA editing. (United States)

    Fukuda, Masatora; Kurihara, Kei; Tanaka, Yasuyoshi; Deshimaru, Masanobu


    Substitutional RNA editing plays a crucial role in the regulation of biological processes. Cleavage of target RNA that depends on the specific site of substitutional RNA editing is a useful tool for analyzing and regulating intracellular processes related to RNA editing. Hammerhead ribozymes have been utilized as small catalytic RNAs for cleaving target RNA at a specific site and may be used for RNA-editing-specific RNA cleavage. Here we reveal a design strategy for a hammerhead ribozyme that specifically recognizes adenosine to inosine (A-to-I) and cytosine to uracil (C-to-U) substitutional RNA-editing sites and cleaves target RNA. Because the hammerhead ribozyme cleaves one base upstream of the target-editing site, the base that pairs with the target-editing site was utilized for recognition. RNA-editing-specific ribozymes were designed such that the recognition base paired only with the edited base. These ribozymes showed A-to-I and C-to-U editing-specific cleavage activity against synthetic serotonin receptor 2C and apolipoprotein B mRNA fragments in vitro, respectively. Additionally, the ribozyme designed for recognizing A-to-I RNA editing at the Q/R site on filamin A (FLNA) showed editing-specific cleavage activity against physiologically edited FLNA mRNA extracted from cells. We demonstrated that our strategy is effective for cleaving target RNA in an editing-dependent manner. The data in this study provided an experimental basis for the RNA-editing-dependent degradation of specific target RNA in vivo.

  12. An investigation of paper based microfluidic devices for size based separation and extraction applications. (United States)

    Zhong, Z W; Wu, R G; Wang, Z P; Tan, H L


    Conventional microfluidic devices are typically complex and expensive. The devices require the use of pneumatic control systems or highly precise pumps to control the flow in the devices. This work investigates an alternative method using paper based microfluidic devices to replace conventional microfluidic devices. Size based separation and extraction experiments conducted were able to separate free dye from a mixed protein and dye solution. Experimental results showed that pure fluorescein isothiocyanate could be separated from a solution of mixed fluorescein isothiocyanate and fluorescein isothiocyanate labeled bovine serum albumin. The analysis readings obtained from a spectrophotometer clearly show that the extracted tartrazine sample did not contain any amount of Blue-BSA, because its absorbance value was 0.000 measured at a wavelength of 590nm, which correlated to Blue-BSA. These demonstrate that paper based microfluidic devices, which are inexpensive and easy to implement, can potentially replace their conventional counterparts by the use of simple geometry designs and the capillary action. These findings will potentially help in future developments of paper based microfluidic devices.

  13. Performance Analysis of Vision-Based Deep Web Data Extraction for Web Document Clustering

    Directory of Open Access Journals (Sweden)

    M. Lavanya


    Full Text Available Web Data Extraction is a critical task by applying various scientific tools and in a broad range of application domains. To extract data from multiple web sites are becoming more obscure, as well to design of web information extraction systems becomes more complex and time-consuming. We also present in this paper so far various risks in web data extraction. Identifying data region from web is a noteworthy crisis for information extraction from the web page. In this paper, performance of vision-based deep web data extraction for web document clustering is presented with experimental result. The proposed approach comprises of two phases: 1 Vision-based web data extraction, where output of phase I is given to second phase and 2 web document clustering. In phase 1, the web page information is segmented into various chunks. From which, surplus noise and duplicate chunks are removed using three parameters, such as hyperlink percentage, noise score and cosine similarity. To identify the relevant chunk, three parameters such as Title word Relevancy, Keyword frequency-based chunk selection, Position features are used and then, a set of keywords are extracted from those main chunks. Finally, the extracted keywords are subjected to web document clustering using Fuzzy c-means clustering (FCM. The experimentation has been performed on two different datasets and the results showed that the proposed VDEC method can achieve stable and good results of about 99.2% and 99.1% precision value in both datasets.

  14. The promise, pitfalls and progress of RNA-interference-based antiviral therapy for respiratory viruses. (United States)

    DeVincenzo, John P


    Advances in the understanding of RNA biological processing and control are leading to new concepts in human therapeutics with practical implications for many human diseases, including antiviral therapy of respiratory viruses. So-called 'non-coding RNA' exerts specific and profound functional control on regulation of protein production and indeed controls the expression of all genes through processes collectively known as RNA interference (RNAi). RNAi is a naturally occurring intracellular process that regulates gene expression through the silencing of specific messenger RNAs (mRNAs). Methods are being developed that allow the catalytic degradation of targeted mRNAs using specifically designed complementary small interfering RNAs (siRNA). siRNAs are now being chemically modified and packaged into advanced delivery systems so as to acquire drug-like properties and the ability to deliver their effects systemically. Recent in vivo studies have provided proofs of the concept that RNAi may be useful therapeutically. Much of the design of these siRNAs can be accomplished bioinformatically, thus potentially expediting drug discovery and opening new avenues of therapy for many uncommon, orphan, or emerging diseases. Theoretically, any disease that can be ameliorated through knockdown of any endogenous or exogenous protein is a potential therapeutic target for RNAi-based therapeutics. Lung diseases in general are attractive targets for RNAi therapeutics, since the location of affected cells increases their accessibility to topical administration of siRNA, and respiratory viral infections are particularly attractive targets for RNAi-based drug discovery and development. RNAi therapeutics have been shown to exert potent antiviral effects against respiratory syncytial virus (RSV), parainfluenza, influenza, coronaviruses, measles and human metapneumoviruses in vitro and in vivo. Recently, a double-blind placebo-controlled clinical trial of an RNAi-based therapeutic against RSV

  15. HTML Extraction Algorithm Based on Property and Data Cell (United States)

    Purnamasari, Detty; Wayan Simri Wicaksana, I.; Harmanto, Suryadi; Yuniar Banowosari, Lintang


    The data available on the Internet is in various models and formats. One form of data representation is a table. Tables extraction is used in process more than one table on the Internet from different sources. Currently the effort is done by using copy-paste that is not automatic process. This article presents an approach to prepare the area, so tables in HTML format can be extracted and converted into a database that make easier to combine the data from many resources. This article was tested on the algorithm 1 used to determine the actual number of columns and rows of the table, as well as algorithm 2 are used to determine the boundary line of the property. Tests conducted at 100 tabular HTML format, and the test results provide the accuracy of the algorithm 1 is 99.9% and the accuracy of the algorithm 2 is 84%.

  16. Extracting Coherent Information from Noise Based Correlation Processing (United States)


    LONG-TERM GOALS The goal of this research is to establish methodologies to utilize ambient noise in the ocean and to determine what scenarios...None PUBLICATIONS [1] “ Monitoring deep-ocean temperatures using acoustic ambinet noise,”K. W. Woolfe, S. Lani, K.G. Sabra, W. A. Kuperman...Geophys. Res. Lett., 42,2878–2884, doi:10.1002/2015GL063438 (2015). [2] “Optimized extraction of coherent arrivals from ambient noise correlations in

  17. Apect-Based Opinion Extraction From Customer Reviews


    Samha, Amani K; Yuefeng Li; Jinglan Zhang


    Text is the main method of communicating informatio n in the digital age. Messages, blogs, news articles, reviews, and opinionated information abounds on the Internet. People commonly purchase products online and post their opinions ab out purchased items. This feedback is displayed publicly to assist others with their purc hasing decisions, creating the need for a mechanism with which to extract and summarize usefu l ...

  18. The Effect of Formaldehyde Fixation on RNA: Optimization of Formaldehyde Adduct Removal



    Formalin-fixed, paraffin-embedded tissues generally provide low yields of extractable RNA that exhibit both covalent modification of nucleic acid bases and strand cleavage. This frustrates efforts to perform retrospective analyses of gene expression using archival tissue specimens. A variety of conditions have been reported to demodify formaldehyde-fixed RNA in different model systems. We studied the reversal of formaldehyde fixation of RNA using a 50 base RNA oligonucleotide and total cellul...

  19. Epitope-tagged protein-based artificial miRNA screens for optimized gene silencing in plants. (United States)

    Li, Jian-Feng; Zhang, Dandan; Sheen, Jen


    Artificial miRNA (amiRNA) technology offers highly specific gene silencing in diverse plant species. The principal challenge in amiRNA application is to select potent amiRNAs from hundreds of bioinformatically designed candidates to enable maximal target gene silencing at the protein level. To address this issue, we developed the epitope-tagged protein-based amiRNA (ETPamir) screens, in which single or multiple potential target genes encoding epitope-tagged proteins are constitutively or inducibly coexpressed with individual amiRNA candidates in plant protoplasts. Accumulation of tagged proteins, detected by immunoblotting with commercial tag antibodies, inversely and quantitatively reflects amiRNA efficacy in vivo. The core procedure, from protoplast isolation to identification of optimal amiRNA, can be completed in 2-3 d. The ETPamir screens circumvent the limited availability of plant antibodies and the complexity of plant amiRNA silencing at target mRNA and/or protein levels. The method can be extended to verify predicted target genes for endogenous plant miRNAs.

  20. New insights into the RNA-based mechanism of action of the anticancer drug 5'-fluorouracil in eukaryotic cells.

    Directory of Open Access Journals (Sweden)

    Laura Mojardín

    Full Text Available 5-Fluorouracil (5FU is a chemotherapeutic drug widely used in treating a range of advanced, solid tumours and, in particular, colorectal cancer. Here, we used high-density tiling DNA microarray technology to obtain the specific transcriptome-wide response induced by 5FU in the eukaryotic model Schizosaccharomyces pombe. This approach combined with real-time quantitative PCR analysis allowed us to detect splicing defects of a significant number of intron-containing mRNA, in addition to identify some rRNA and tRNA processing defects after 5FU treatment. Interestingly, our studies also revealed that 5FU specifically induced the expression of certain genes implicated in the processing of mRNA, tRNA and rRNA precursors, and in the post-transcriptional modification of uracil residues in RNA. The transcription of several tRNA genes was also significantly induced after drug exposure. These transcriptional changes might represent a cellular response mechanism to counteract 5FU damage since deletion strains for some of these up-regulated genes were hypersensitive to 5FU. Moreover, most of these RNA processing genes have human orthologs that participate in conserved pathways, suggesting that they could be novel targets to improve the efficacy of 5FU-based treatments.

  1. A green deep eutectic solvent-based aqueous two-phase system for protein extracting

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Kaijia; Wang, Yuzhi, E-mail:; Huang, Yanhua; Li, Na; Wen, Qian


    Highlights: • A strategy for the protein purification with a deep eutectic solvent(DES)-based aqueous two-phase system. • Choline chloride-glycerin DES was selected as the extraction solvent. • Bovine serum albumin and trypsin were used as the analytes. • Aggregation phenomenon was detected in the mechanism research. - Abstract: As a new type of green solvent, deep eutectic solvent (DES) has been applied for the extraction of proteins with an aqueous two-phase system (ATPS) in this work. Four kinds of choline chloride (ChCl)-based DESs were synthesized to extract bovine serum albumin (BSA), and ChCl-glycerol was selected as the suitable extraction solvent. Single factor experiments have been done to investigate the effects of the extraction process, including the amount of DES, the concentration of salt, the mass of protein, the shaking time, the temperature and PH value. Experimental results show 98.16% of the BSA could be extracted into the DES-rich phase in a single-step extraction under the optimized conditions. A high extraction efficiency of 94.36% was achieved, while the conditions were applied to the extraction of trypsin (Try). Precision, repeatability and stability experiments were studied and the relative standard deviations (RSD) of the extraction efficiency were 0.4246% (n = 3), 1.6057% (n = 3) and 1.6132% (n = 3), respectively. Conformation of BSA was not changed during the extraction process according to the investigation of UV–vis spectra, FT-IR spectra and CD spectra of BSA. The conductivity, dynamic light scattering (DLS) and transmission electron microscopy (TEM) were used to explore the mechanism of the extraction. It turned out that the formation of DES–protein aggregates play a significant role in the separation process. All the results suggest that ChCl-based DES-ATPS are supposed to have the potential to provide new possibilities in the separation of proteins.

  2. PCR-based diversity estimates of artificial and environmental 18S rRNA gene libraries. (United States)

    Potvin, Marianne; Lovejoy, Connie


    Environmental clone libraries constructed using small subunit ribosomal RNA (rRNA) or other gene-specific primers have become the standard molecular approach for identifying microorganisms directly from their environment. This technique includes an initial polymerase chain reaction (PCR) amplification step of a phylogenetically useful marker gene using universal primers. Although it is acknowledged that such primers introduce biases, there have been few studies if any to date systematically examining such bias in eukaryotic microbes. We investigated some implications of such bias by constructing clone libraries using several universal primer pairs targeting rRNA genes. Firstly, we constructed artificial libraries using a known mix of small cultured pelagic arctic algae with representatives from five major lineages and secondly we investigated environmental samples using several primer pairs. No primer pair retrieved all of the original algae in the artificial clone libraries and all showed a favorable bias toward the dinoflagellate Polarella glacialis and a bias against the prasinophyte Micromonas and a pennate diatom. Several other species were retrieved by only one primer pair tested. Despite this, sequences from nine environmental libraries were diverse and contained representatives from all major eukaryotic clades expected in marine samples. Further, libraries from the same sample grouped together using Bray-Curtis clustering, irrespective of primer pairs. We conclude that environmental PCR-based techniques are sufficient to compare samples, but the total diversity will probably always be underestimated and relative abundance estimates should be treated with caution.

  3. The Automatic Generation of Chinese Outline Font Based on Stroke Extraction

    Institute of Scientific and Technical Information of China (English)


    A new method to obtain spline outline description of Chinese font based on stroke extraction is presented.It has two primary advantages:(1)the quality of Chinese output is greatly improved;(2)the memory requirement is reduced.The method for stroke extraction is discussed in detail and experimental results are presented.

  4. Secondary Structure Predictions for Long RNA Sequences Based on Inversion Excursions and MapReduce. (United States)

    Yehdego, Daniel T; Zhang, Boyu; Kodimala, Vikram K R; Johnson, Kyle L; Taufer, Michela; Leung, Ming-Ying


    Secondary structures of ribonucleic acid (RNA) molecules play important roles in many biological processes including gene expression and regulation. Experimental observations and computing limitations suggest that we can approach the secondary structure prediction problem for long RNA sequences by segmenting them into shorter chunks, predicting the secondary structures of each chunk individually using existing prediction programs, and then assembling the results to give the structure of the original sequence. The selection of cutting points is a crucial component of the segmenting step. Noting that stem-loops and pseudoknots always contain an inversion, i.e., a stretch of nucleotides followed closely by its inverse complementary sequence, we developed two cutting methods for segmenting long RNA sequences based on inversion excursions: the centered and optimized method. Each step of searching for inversions, chunking, and predictions can be performed in parallel. In this paper we use a MapReduce framework, i.e., Hadoop, to extensively explore meaningful inversion stem lengths and gap sizes for the segmentation and identify correlations between chunking methods and prediction accuracy. We show that for a set of long RNA sequences in the RFAM database, whose secondary structures are known to contain pseudoknots, our approach predicts secondary structures more accurately than methods that do not segment the sequence, when the latter predictions are possible computationally. We also show that, as sequences exceed certain lengths, some programs cannot computationally predict pseudoknots while our chunking methods can. Overall, our predicted structures still retain the accuracy level of the original prediction programs when compared with known experimental secondary structure.

  5. Transcriptomics in the tropics: Total RNA-based profiling of Costa Rican bromeliad-associated communities

    Directory of Open Access Journals (Sweden)

    Shana K. Goffredi


    Full Text Available RNA-Seq was used to examine the microbial, eukaryotic, and viral communities in water catchments (‘tanks’ formed by tropical bromeliads from Costa Rica. In total, transcripts with taxonomic affiliation to a wide array of bacteria, archaea, and eukaryotes, were observed, as well as RNA-viruses that appeared related to the specific presence of eukaryotes. Bacteria from 25 phyla appeared to comprise the majority of transcripts in one tank (Wg24, compared to only 14 phyla in the other (Wg25. Conversely, eukaryotes from only 16 classes comprised the majority of transcripts in Wg24, compared to 24 classes in the Wg25, revealing a greater eukaryote diversity in the latter. Given that these bromeliads had tanks of similar size (i.e. vertical oxygen gradient, and were neighboring with presumed similar light regime and acquisition of leaf litter through-fall, it is possible that pH was the factor governing these differences in bacterial and eukaryotic communities (Wg24 had a tank pH of 3.6 and Wg25 had a tank pH of 6.2. Archaeal diversity was similar in both tanks, represented by 7 orders, with the exception of Methanocellales transcripts uniquely recovered from Wg25. Based on measures of FPKG (fragments mapped per kilobase of gene length, genes involved in methanogenesis, in addition to a spirochaete flagellin gene, were among those most highly expressed in Wg25. Conversely, aldehyde dehydrogenase and monosaccharide-binding protein were among genes most highly expressed in Wg24. The ability to observe specific presence of insect, plant, and fungi-associated RNA-viruses was unexpected. As with other techniques, there are inherent biases in the use of RNA-Seq, however, these data suggest the possibility of understanding the entire community, including ecological interactions, via simultaneous analysis of microbial, eukaryotic, and viral transcripts.

  6. Transcriptomics in the tropics: Total RNA-based profiling of Costa Rican bromeliad-associated communities. (United States)

    Goffredi, Shana K; Jang, Gene E; Haroon, Mohamed F


    RNA-Seq was used to examine the microbial, eukaryotic, and viral communities in water catchments ('tanks') formed by tropical bromeliads from Costa Rica. In total, transcripts with taxonomic affiliation to a wide array of bacteria, archaea, and eukaryotes, were observed, as well as RNA-viruses that appeared related to the specific presence of eukaryotes. Bacteria from 25 phyla appeared to comprise the majority of transcripts in one tank (Wg24), compared to only 14 phyla in the other (Wg25). Conversely, eukaryotes from only 16 classes comprised the majority of transcripts in Wg24, compared to 24 classes in the Wg25, revealing a greater eukaryote diversity in the latter. Given that these bromeliads had tanks of similar size (i.e. vertical oxygen gradient), and were neighboring with presumed similar light regime and acquisition of leaf litter through-fall, it is possible that pH was the factor governing these differences in bacterial and eukaryotic communities (Wg24 had a tank pH of 3.6 and Wg25 had a tank pH of 6.2). Archaeal diversity was similar in both tanks, represented by 7 orders, with the exception of Methanocellales transcripts uniquely recovered from Wg25. Based on measures of FPKG (fragments mapped per kilobase of gene length), genes involved in methanogenesis, in addition to a spirochaete flagellin gene, were among those most highly expressed in Wg25. Conversely, aldehyde dehydrogenase and monosaccharide-binding protein were among genes most highly expressed in Wg24. The ability to observe specific presence of insect, plant, and fungi-associated RNA-viruses was unexpected. As with other techniques, there are inherent biases in the use of RNA-Seq, however, these data suggest the possibility of understanding the entire community, including ecological interactions, via simultaneous analysis of microbial, eukaryotic, and viral transcripts.

  7. Edge-Based Feature Extraction Method and Its Application to Image Retrieval

    Directory of Open Access Journals (Sweden)

    G. Ohashi


    Full Text Available We propose a novel feature extraction method for content-bases image retrieval using graphical rough sketches. The proposed method extracts features based on the shape and texture of objects. This edge-based feature extraction method functions by representing the relative positional relationship between edge pixels, and has the advantage of being shift-, scale-, and rotation-invariant. In order to verify its effectiveness, we applied the proposed method to 1,650 images obtained from the Hamamatsu-city Museum of Musical Instruments and 5,500 images obtained from Corel Photo Gallery. The results verified that the proposed method is an effective tool for achieving accurate retrieval.

  8. The long and short of antiviral defense: small RNA-based immunity in insects

    NARCIS (Netherlands)

    Bronkhorst, A.W.; Rij, R.P. van


    The host RNA interference (RNAi) pathway of insects senses virus infection and induces an antiviral response to restrict virus replication. Dicer-2 detects viral double-stranded RNA, produced by RNA and DNA viruses, and generates viral small interfering RNAs (vsiRNAs). Recent small RNA profiling stu

  9. Fusion of Pixel-based and Object-based Features for Road Centerline Extraction from High-resolution Satellite Imagery

    Directory of Open Access Journals (Sweden)

    CAO Yungang


    Full Text Available A novel approach for road centerline extraction from high spatial resolution satellite imagery is proposed by fusing both pixel-based and object-based features. Firstly, texture and shape features are extracted at the pixel level, and spectral features are extracted at the object level based on multi-scale image segmentation maps. Then, extracted multiple features are utilized in the fusion framework of Dempster-Shafer evidence theory to roughly identify the road network regions. Finally, an automatic noise removing algorithm combined with the tensor voting strategy is presented to accurately extract the road centerline. Experimental results using high-resolution satellite imageries with different scenes and spatial resolutions showed that the proposed approach compared favorably with the traditional methods, particularly in the aspect of eliminating the salt noise and conglutination phenomenon.

  10. Evaluation of DNA Extraction Methods Suitable for PCR-based Detection and Genotyping of Clostridium botulinum

    DEFF Research Database (Denmark)

    Auricchio, Bruna; Anniballi, Fabrizio; Fiore, Alfonsina


    Sufficient quality and quantity of extracted DNA is critical to detecting and performing genotyping of Clostridium botulinum by means of PCR-based methods. An ideal extraction method has to optimize DNA yield, minimize DNA degradation, allow multiple samples to be extracted, and be efficient...... in terms of cost, time, labor, and supplies. Eleven botulinum toxin–producing clostridia strains and 25 samples (10 food, 13 clinical, and 2 environmental samples) naturally contaminated with botulinum toxin–producing clostridia were used to compare 4 DNA extraction procedures: Chelex® 100 matrix, Phenol......-Cloroform-Isoamyl alcohol, NucliSENS® magnetic extraction kit, and DNeasy® Blood & Tissue kit. Integrity, purity, and amount of amplifiable DNA were evaluated. The results show that the DNeasy® Blood & Tissue kit is the best extraction method evaluated because it provided the most pure, intact, and amplifiable DNA. However...

  11. Hardware Prototyping of Neural Network based Fetal Electrocardiogram Extraction (United States)

    Hasan, M. A.; Reaz, M. B. I.


    The aim of this paper is to model the algorithm for Fetal ECG (FECG) extraction from composite abdominal ECG (AECG) using VHDL (Very High Speed Integrated Circuit Hardware Description Language) for FPGA (Field Programmable Gate Array) implementation. Artificial Neural Network that provides efficient and effective ways of separating FECG signal from composite AECG signal has been designed. The proposed method gives an accuracy of 93.7% for R-peak detection in FHR monitoring. The designed VHDL model is synthesized and fitted into Altera's Stratix II EP2S15F484C3 using the Quartus II version 8.0 Web Edition for FPGA implementation.

  12. Subpixel accuracy for extracting groove center based on corner detection

    Institute of Scientific and Technical Information of China (English)

    Liu Suyi; Wang Guorong; Shi Yonghua


    Subpixel accuracy for V-groove center in robot welding is researched and a software measure to increase the accuracy of seam tracking by laser is presented.LOG( Laplacian of Gaussian ) operator is adopted to detect image edge.Vgroove center is extracted by corner detection of extremum curvature.Subpixel position is obtained by Lagarange polynomial interpolation algorithm.Experiment results show that the method is brief and applied, and is sufficient for the real time of robot welding by laser sensors.

  13. Fragment-based discovery of hepatitis C virus NS5b RNA polymerase inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Antonysamy, Stephen S.; Aubol, Brandon; Blaney, Jeff; Browner, Michelle F.; Giannetti, Anthony M.; Harris, Seth F.; Hébert, Normand; Hendle, Jörg; Hopkins, Stephanie; Jefferson, Elizabeth; Kissinger, Charles; Leveque, Vincent; Marciano, David; McGee, Ethel; Nájera, Isabel; Nolan, Brian; Tomimoto, Masaki; Torres, Eduardo; Wright, Tobi (SGX); (Roche)


    Non-nucleoside inhibitors of HCV NS5b RNA polymerase were discovered by a fragment-based lead discovery approach, beginning with crystallographic fragment screening. The NS5b binding affinity and biochemical activity of fragment hits and inhibitors was determined by surface plasmon resonance (Biacore) and an enzyme inhibition assay, respectively. Crystallographic fragment screening hits with {approx}1-10 mM binding affinity (K{sub D}) were iteratively optimized to give leads with {approx}200 nM biochemical activity and low {micro}M cellular activity in a Replicon assay.

  14. From conventional therapy toward microRNA-based therapy in acute promyelocytic leukemia (United States)

    Ehtesham, Naeim; Sharifi, Mohammadreza


    Acute promyelocytic leukemia (APL) is a hematopoietic malignancy that is known with its special cytogenetic feature. Several studies have surveyed expression signature of microRNAs (miRNAs) in APL patients, especially patients who are treated with conventional therapy of this disease. Using miRNAs as diagnostic or prognostic biomarkers in various cancers has been widely studied. Currently, most studies are focusing on exploiting miRNAs as therapeutic tools, and promising progress has been achieved in this field. Recently, studies in the field of miRNA-based therapy in APL have been started. PMID:28028527

  15. Continuous cell electroporation for efficient DNA and siRNA delivery based on laminar microfluidic chips. (United States)

    Wei, Zewen; Li, Zhihong


    Electroporation is a high-efficiency and low-toxicity physical gene transfer method. Traditional electroporation is limited to only low volume cell samples. Here we present a continuous cell electroporation method based on commonly used microfluidic chip fabrication technology. Using easily fabricated PDMS microfluidic chip, syringe pumps, and pulse generator, we show efficient delivery of both DNA and siRNA into different cell lines. We describe the protocol of chip fabrication, apparatus setup, and cell electroporation assay. Typically, the fabrication of the devices takes 1 or 2 days and the continuous electroporation assay takes 1 h.

  16. Hexafluoroisopropanol-induced catanionic-surfactants-based coacervate extraction for analysis of lysozyme. (United States)

    Xu, Jia; Niu, Manli; Xiao, Yuxiu


    A coacervate extraction method, based on hexafluoroisopropanol (HFIP)-induced catanionic surfactants and coupled with a back-extraction procedure, was developed for separation and purification of proteins, using sodium dodecyl sulfate (SDS) and dodecyltrimethyl ammonium bromide (DTAB) as representative catanionic surfactants and lysozyme as a model protein. After the coacervate extraction and back extraction, the obtained lysozyme solutions were examined in terms of quantitative analysis by capillary electrophoresis, bacteriolytic activity, and circular dichroism (CD). The effects of several parameters including back-extraction solvent, HFIP content, total surfactant concentration, and SDS/DTAB molar ratio were investigated in detail on the extraction efficiency and activity of lysozyme. Under the optimized extraction conditions (66 mM KH2PO4 buffer with pH 6.2 as back-extraction solvent, SDS/DTAB molar ratio = 1:1 mol/mol, total surfactant concentration = 30 mM, HIFP concentration = 8 % v/v), the extraction recovery was 89.8 % (±4.7, n = 3), limit of detection was 2.2 (±0.3, n = 3) μg mL(-1), and meanwhile nearly 65 % of native lysozyme activity was retained. In addition, the activity and CD assays showed that SDS/DTAB molar ratio had a significant influence on the activity and structure of lysozyme after extraction. The DTAB-rich extraction systems, in which the DTAB mole fraction was equal to or larger than 70 %, could keep the activity and structure of lysozyme almost in the native state. Graphical Abstract Procedure of HFIP-induced SDS/DTAB coacervate extraction and back extraction of lysozyme.


    Institute of Scientific and Technical Information of China (English)

    Zheng Shuxian; Zhao Wanhua; Lu Bingheng; Zhao Zhao


    In the prosthetic socket design, aimed at the high cost and radiation deficiency caused by CT scanning which is a routine technique to obtain the cross-sectional image of the residual limb, a new ultrasonic scanning method is developed to acquire the bones and skin contours of the residual limb. Using a pig fore-leg as the scanning object, an overlapping algorithm is designed to reconstruct the 2D cross-sectional image, the contours of the bone and skin are extracted using edge detection algorithm and the 3D model of the pig fore-leg is reconstructed by using reverse engineering technology. The results of checking the accuracy of the image by scanning a cylinder work pieces show that the extracted contours of the cylinder are quite close to the standard circumference. So it is feasible to get the contours of bones and skin by ultrasonic scanning. The ultrasonic scanning system featuring no radiation and low cost is a kind of new means of cross section scanning for medical images.

  18. The P3 domain of eukaryotic RNases P/MRP: making a protein-rich RNA-based enzyme. (United States)

    Perederina, Anna; Krasilnikov, Andrey S


    Nuclear Ribonuclease (RNase) P is a universal essential RNA-based enzyme made of a catalytic RNA component and a protein part; eukaryotic RNase P is closely related to a universal eukaryotic ribonucleoprotein RNase MRP. The protein part of the eukaryotic RNases P/MRP is dramatically more complex than that in bacterial and archaeal RNases P. The increase in the complexity of the protein part in eukaryotic RNases P/MRP was accompanied by the appearance of a novel structural element in the RNA component: an essential and phylogenetically conserved helix-loop-helix P3 RNA domain. The crystal structure of the P3 RNA domain in a complex with protein components Pop6 and Pop7 has been recently solved. Here we discuss the most salient structural features of the P3 domain as well as its possible role in the evolutionary transition to the protein-rich eukaryotic RNases P/MRP.

  19. RNA. Introduction. (United States)

    Bao, Marie Z; Kruger, Robert P; Rivas, Fabiola; Smith, Orla; Szewczak, Lara


    Two scientists walk into a bar. After a pint and an exchange of pleasantries, one says to the other, "Where do you come from? Scientifically, I mean." The queried scientist responds, "Out of the RNA world." "Don't we all," the asker responds chuckling. Fifteen years ago, the joke would have been made with a nod to the notion that life arose from an RNA-based precursor, the so-called "RNA world." Yet had this conversation happened last week, the scientists would also be grinning in appreciation of the extent to which contemporary cellular biology is steeped in all things RNA. Ours is truly an RNA world.In this year's special review issue, the Cell editorial team has brought together articles focused on RNA in the modern world, providing perspectives on classical and emerging areas of inquiry. We extend our thanks to the many distinguished experts who contributed their time and effort as authors and reviewers to make the issue informative, thought-provoking, and timely. We hope that this collection of articles, written as we stand on the verge of a new wave of RNA biology, edifies and inspires by revealing the inner workings of these versatile molecules and by highlighting the next key questions that need to be addressed as we strive to understand the full functional scope of RNA in cells.

  20. Efficient delivery of Notch1 siRNA to SKOV3 cells by cationic cholesterol derivative-based liposome (United States)

    Zhao, Yun-Chun; Zhang, Li; Feng, Shi-Sen; Hong, Lu; Zheng, Hai-Li; Chen, Li-Li; Zheng, Xiao-Ling; Ye, Yi-Qing; Zhao, Meng-Dan; Wang, Wen-Xi; Zheng, Cai-Hong


    A novel cationic cholesterol derivative-based small interfering RNA (siRNA) interference strategy was suggested to inhibit Notch1 activation in SKOV3 cells for the gene therapy of ovarian cancer. The cationic cholesterol derivative, N-(cholesterylhemisuccinoyl-amino-3-propyl)-N, N-dimethylamine (DMAPA-chems) liposome, was incubated with siRNA at different nitrogen-to-phosphate ratios to form stabilized, near-spherical siRNA/DMAPA-chems nanoparticles with sizes of 100–200 nm and zeta potentials of 40–50 mV. The siRNA/DMAPA-chems nanoparticles protected siRNA from nuclease degradation in 25% fetal bovine serum. The nanoparticles exhibited high cell uptake and Notch1 gene knockdown efficiency in SKOV3 cells at an nitrogen-to-phosphate ratio of 100 and an siRNA concentration of 50 nM. They also inhibited the growth and promoted the apoptosis of SKOV3 cells. These results may provide the potential for using cationic cholesterol derivatives as efficient nonviral siRNA carriers for the suppression of Notch1 activation in ovarian cancer cells.

  1. Texture based feature extraction methods for content based medical image retrieval systems. (United States)

    Ergen, Burhan; Baykara, Muhammet


    The developments of content based image retrieval (CBIR) systems used for image archiving are continued and one of the important research topics. Although some studies have been presented general image achieving, proposed CBIR systems for archiving of medical images are not very efficient. In presented study, it is examined the retrieval efficiency rate of spatial methods used for feature extraction for medical image retrieval systems. The investigated algorithms in this study depend on gray level co-occurrence matrix (GLCM), gray level run length matrix (GLRLM), and Gabor wavelet accepted as spatial methods. In the experiments, the database is built including hundreds of medical images such as brain, lung, sinus, and bone. The results obtained in this study shows that queries based on statistics obtained from GLCM are satisfied. However, it is observed that Gabor Wavelet has been the most effective and accurate method.

  2. RNA extracted from blood samples with a rapid automated procedure is fit for molecular diagnosis or minimal residual disease monitoring in patients with a variety of malignant blood disorders. (United States)

    Bechlian, Didier; Honstettre, Amélie; Terrier, Michèle; Brest, Christelle; Malenfant, Carine; Mozziconacci, Marie-Joëlle; Chabannon, Christian


    Scientific studies in oncology, cancer diagnosis, and monitoring tumor response to therapeutics currently rely on a growing number of clinico-pathological information. These often include molecular analyses. The quality of these analyses depends on both pre-analytical and analytical information and often includes the extraction of DNA and/or RNA from human tissues and cells. The quality and quantity of obtained nucleic acids are of utmost importance. The use of automated techniques presents several advantages over manual techniques, such as reducing technical time and thus cost, and facilitating standardization. The purpose of this study was to validate an automated technique for RNA extraction from cells of patients treated for various malignant blood diseases. A well-established manual technique was compared to an automated technique, in order to extract RNA from blood samples drawn for the molecular diagnosis of a variety of leukemic diseases or monitoring of minimal residual disease. The quality of the RNA was evaluated by real-time quantitative RT-PCR (RQ-PCR) analyses of the Abelson gene transcript. The results show that both techniques produce RNA with comparable quality and quantity, thus suggesting that an automated technique can be substituted for the reference and manual technique used in the daily routine of a molecular pathology laboratory involved in minimal residual disease monitoring. Increased costs of reagents and disposables used for automated techniques can be compensated by a decrease in human resource.

  3. A Shortest Dependency Path Based Convolutional Neural Network for Protein-Protein Relation Extraction



    The state-of-the-art methods for protein-protein interaction (PPI) extraction are primarily based on kernel methods, and their performances strongly depend on the handcraft features. In this paper, we tackle PPI extraction by using convolutional neural networks (CNN) and propose a shortest dependency path based CNN (sdpCNN) model. The proposed method (1) only takes the sdp and word embedding as input and (2) could avoid bias from feature selection by using CNN. We performed experiments on sta...

  4. Feature evaluation and extraction based on neural network in analog circuit fault diagnosis

    Institute of Scientific and Technical Information of China (English)

    Yuan Haiying; Chen Guangju; Xie Yongle


    Choosing the right characteristic parameter is the key to fault diagnosis in analog circuit.The feature evaluation and extraction methods based on neural network are presented.Parameter evaluation of circuit features is realized by training results from neural network; the superior nonlinear mapping capability is competent for extracting fault features which are normalized and compressed subsequently.The complex classification problem on fault pattern recognition in analog circuit is transferred into feature processing stage by feature extraction based on neural network effectively, which improves the diagnosis efficiency.A fault diagnosis illustration validated this method.

  5. Feature Fusion Based Road Extraction for HJ-1-C SAR Image


    Lu Ping-ping; Du Kang-ning; Yu Wei-dong; Wang Yu; Deng Yun-kai


    Road network extraction in SAR images is one of the key tasks of military and civilian technologies. To solve the issues of road extraction of HJ-1-C SAR images, a road extraction algorithm is proposed based on the integration of ratio and directional information. Due to the characteristic narrow dynamic range and low signal to noise ratio of HJ-1-C SAR images, a nonlinear quantization and an image filtering method based on a multi-scale autoregressive model are proposed here. A road extracti...

  6. Clinical performance of the new Roche COBAS (R) TaqMan HCV test and high pure system for extraction, detection and quantitation of HCV RNA in plasma and serum

    NARCIS (Netherlands)

    H.C. Gelderblom; S. Menting; M.G. Beld


    We evaluated the Roche COBAS (R) TaqMan HCV Test For Use With The High Pure System (TaqMan HPS; Roche Diagnostics), for the extraction, detection and quantitation of hepatitis C virus (HCV) RNA in serum or plasma of HCV-infected individuals. The TaqMan HPS is a real-time PCR assay with a reported li

  7. Effect of polysaccharides extract of rhizoma atractylodis macrocephalae on thymus, spleen and cardiac indexes, caspase-3 activity ratio, Smac/DIABLO and HtrA2/Omi protein and mRNA expression levels in aged rats. (United States)

    Guo, Ling; Sun, Yong Le; Wang, Ai Hong; Xu, Chong En; Zhang, Meng Yuan


    This study was designed to determine the possible protective effect of polysaccharides extract of rhizoma atractylodis macrocephalae on heart function in aged rats. Polysaccharides extract of rhizoma atractylodis macrocephalae was administered to aged rats. Results showed that thymus, spleen and cardiac indexs were significantly increased, whereas caspase-3 activity ratio, Smac/DIABLO and HtrA2/Omi protein expression, Smac/DIABLO and HtrA2/Omi mRNA expression levels were markedly reduced. It can be concluded that polysaccharides extract of rhizoma atractylodis macrocephalae may enhance immunity and improve heart function in aged rats.

  8. Sorting live stem cells based on Sox2 mRNA expression.

    Directory of Open Access Journals (Sweden)

    Hans M Larsson

    Full Text Available While cell sorting usually relies on cell-surface protein markers, molecular beacons (MBs offer the potential to sort cells based on the presence of any expressed mRNA and in principle could be extremely useful to sort rare cell populations from primary isolates. We show here how stem cells can be purified from mixed cell populations by sorting based on MBs. Specifically, we designed molecular beacons targeting Sox2, a well-known stem cell marker for murine embryonic (mES and neural stem cells (NSC. One of our designed molecular beacons displayed an increase in fluorescence compared to a nonspecific molecular beacon both in vitro and in vivo when tested in mES and NSCs. We sorted Sox2-MB(+SSEA1(+ cells from a mixed population of 4-day retinoic acid-treated mES cells and effectively isolated live undifferentiated stem cells. Additionally, Sox2-MB(+ cells isolated from primary mouse brains were sorted and generated neurospheres with higher efficiency than Sox2-MB(- cells. These results demonstrate the utility of MBs for stem cell sorting in an mRNA-specific manner.

  9. Designing RNA-based genetic control systems for efficient production from engineered metabolic pathways. (United States)

    Stevens, Jason T; Carothers, James M


    Engineered metabolic pathways can be augmented with dynamic regulatory controllers to increase production titers by minimizing toxicity and helping cells maintain homeostasis. We investigated the potential for dynamic RNA-based genetic control systems to increase production through simulation analysis of an engineered p-aminostyrene (p-AS) pathway in E. coli. To map the entire design space, we formulated 729 unique mechanistic models corresponding to all of the possible control topologies and mechanistic implementations in the system under study. Two thousand sampled simulations were performed for each of the 729 system designs to relate the potential effects of dynamic control to increases in p-AS production (total of 3 × 10(6) simulations). Our analysis indicates that dynamic control strategies employing aptazyme-regulated expression devices (aREDs) can yield >10-fold improvements over static control. We uncovered generalizable trends in successful control architectures and found that highly performing RNA-based control systems are experimentally tractable. Analyzing the metabolic control state space to predict optimal genetic control strategies promises to enhance the design of metabolic pathways.

  10. A Method of Road Extraction from High-resolution Remote Sensing Images Based on Shape Features

    Directory of Open Access Journals (Sweden)

    LEI Xiaoqi


    Full Text Available Road extraction from high-resolution remote sensing image is an important and difficult task.Since remote sensing images include complicated information,the methods that extract roads by spectral,texture and linear features have certain limitations.Also,many methods need human-intervention to get the road seeds(semi-automatic extraction,which have the great human-dependence and low efficiency.The road-extraction method,which uses the image segmentation based on principle of local gray consistency and integration shape features,is proposed in this paper.Firstly,the image is segmented,and then the linear and curve roads are obtained by using several object shape features,so the method that just only extract linear roads are rectified.Secondly,the step of road extraction is carried out based on the region growth,the road seeds are automatic selected and the road network is extracted.Finally,the extracted roads are regulated by combining the edge information.In experiments,the images that including the better gray uniform of road and the worse illuminated of road surface were chosen,and the results prove that the method of this study is promising.

  11. Extraction of proteins with ionic liquid aqueous two-phase system based on guanidine ionic liquid. (United States)

    Zeng, Qun; Wang, Yuzhi; Li, Na; Huang, Xiu; Ding, Xueqin; Lin, Xiao; Huang, Songyun; Liu, Xiaojie


    Eight kinds of green ionic liquids were synthesized, and an ionic liquid aqueous two-phase system (ILATPS) based on 1,1,3,3-tetramethylguandine acrylate (TMGA) guanidine ionic liquid was first time studied for the extraction of proteins. Single factor experiments proved that the extraction efficiency of bovine serum albumin (BSA) was influenced by the mass of IL, K2HPO4 and BSA, also related to the separation time and temperature. The optimum conditions were determined through orthogonal experiment by the five factors described above. The results showed that under the optimum conditions, the extraction efficiency could reach up to 99.6243%. The relative standard deviations (RSD) of extraction efficiencies in precision experiment, repeatability experiment and stability experiment were 0.8156% (n=5), 1.6173% (n=5) and 1.6292% (n=5), respectively. UV-vis and FT-IR spectra confirmed that there were no chemical interactions between BSA and ionic liquid in the extraction process, and the conformation of the protein was not changed after extraction. The conductivity, DLS and TEM were combined to investigate the microstructure of the top phase and the possible mechanism for the extraction. The results showed that hydrophobic interaction, hydrogen bonding interaction and the salt out effect played important roles in the transferring process, and the aggregation and embrace phenomenon was the main driving force for the separation. All these results proved that guanidine ionic liquid-based ATPSs have the potential to offer new possibility in the extraction of proteins.

  12. Effects of single-base substitutions within the acanthamoeba castellanii rRNA promoter on transcription and on binding of transcription initiation factor and RNA polymerase I

    Energy Technology Data Exchange (ETDEWEB)

    Kownin, P.; Bateman, E.; Paule, M.R.


    Single-point mutations were introduced into the promoter region of the Acanthamoeba castellanii rRNA gene by chemical mutagen treatment of a single-stranded clone in vitro, followed by reverse transcription and cloning of the altered fragment. The promoter mutants were tested for transcription initiation factor (TIF) binding by a template commitment assay plus DNase I footprinting and for transcription by an in vitro runoff assay. Point mutations within the previously identified TIF interaction region (between -20 and -47, motifs A and B) indicated that TIF interacts most strongly with a sequence centered at -29 and less tightly with sequences upstream and downstream. Some alterations of the base sequence closer to the transcription start site (and outside the TIF-protected site) also significantly decrease specific RNA synthesis in vitro. These were within the region which is protected from DNAse I digestion by polymerase I, but these mutations did not detectably affect the binding of polymerase to the promoter.

  13. 16S rRNA-based detection of oral pathogens in coronary atherosclerotic plaque

    Directory of Open Access Journals (Sweden)

    Mahendra Jaideep


    Full Text Available Background: Atherosclerosis develops as a response of the vessel wall to injury. Chronic bacterial infections have been associated with an increased risk for atherosclerosis and coronary artery disease. The ability of oral pathogens to colonize in coronary atheromatous plaque is well known. Aim: The aim of this study was to detect the presence of Treponema denticola, Porphyromonas gingivalis and Campylobacter rectus in the subgingival and atherosclerotic plaques of patients with coronary artery disease. Materials and Methods: Fifty-one patients in the age group of 40-80 years with coronary artery disease were selected for the study. DNA was extracted from the plaque samples. The specific primers for T. denticola, C. rectus and P. gingivalis were used to amplify a part of the 16S rRNA gene by polymerase chain reaction. Statistical Analysis Used: Chi-square analysis, correlation coefficient and prevalence percentage of the microorganisms were carried out for the analysis. Results: Of the 51 patients, T. denticola, C. rectus and P. gingivalis were detected in 49.01%, 21.51% and 45.10% of the atherosclerotic plaque samples. Conclusions: Our study revealed the presence of bacterial DNA of the oral pathogenic microorganisms in coronary atherosclerotic plaques. The presence of the bacterial DNA in the coronary atherosclerotic plaques in significant proportion may suggest the possible relationship between periodontal bacterial infection and genesis of coronary atherosclerosis.

  14. Point of care nucleic acid detection of viable pathogenic bacteria with isothermal RNA amplification based paper biosensor (United States)

    Liu, Hongxing; Xing, Da; Zhou, Xiaoming


    Food-borne pathogens such as Listeria monocytogenes have been recognized as a major cause of human infections worldwide, leading to substantial health problems. Food-borne pathogen identification needs to be simpler, cheaper and more reliable than the current traditional methods. Here, we have constructed a low-cost paper biosensor for the detection of viable pathogenic bacteria with the naked eye. In this study, an effective isothermal amplification method was used to amplify the hlyA mRNA gene, a specific RNA marker in Listeria monocytogenes. The amplification products were applied to the paper biosensor to perform a visual test, in which endpoint detection was performed using sandwich hybridization assays. When the RNA products migrated along the paper biosensor by capillary action, the gold nanoparticles accumulated at the designated Test line and Control line. Under optimized experimental conditions, as little as 0.5 pg/μL genomic RNA from Listeria monocytogenes could be detected. The whole assay process, including RNA extraction, amplification, and visualization, can be completed within several hours. The developed method is suitable for point-of-care applications to detect food-borne pathogens, as it can effectively overcome the false-positive results caused by amplifying nonviable Listeria monocytogenes.

  15. An analytical platform for mass spectrometry-based identification and chemical analysis of RNA in ribonucleoprotein complexes. (United States)

    Taoka, Masato; Yamauchi, Yoshio; Nobe, Yuko; Masaki, Shunpei; Nakayama, Hiroshi; Ishikawa, Hideaki; Takahashi, Nobuhiro; Isobe, Toshiaki


    We describe here a mass spectrometry (MS)-based analytical platform of RNA, which combines direct nano-flow reversed-phase liquid chromatography (RPLC) on a spray tip column and a high-resolution LTQ-Orbitrap mass spectrometer. Operating RPLC under a very low flow rate with volatile solvents and MS in the negative mode, we could estimate highly accurate mass values sufficient to predict the nucleotide composition of a approximately 21-nucleotide small interfering RNA, detect post-transcriptional modifications in yeast tRNA, and perform collision-induced dissociation/tandem MS-based structural analysis of nucleolytic fragments of RNA at a sub-femtomole level. Importantly, the method allowed the identification and chemical analysis of small RNAs in ribonucleoprotein (RNP) complex, such as the pre-spliceosomal RNP complex, which was pulled down from cultured cells with a tagged protein cofactor as bait. We have recently developed a unique genome-oriented database search engine, Ariadne, which allows tandem MS-based identification of RNAs in biological samples. Thus, the method presented here has broad potential for automated analysis of RNA; it complements conventional molecular biology-based techniques and is particularly suited for simultaneous analysis of the composition, structure, interaction, and dynamics of RNA and protein components in various cellular RNP complexes.

  16. Validation of artificial microRNA expression by poly(A) tailing-based RT-PCR




    Authors: Rui Shi, Chenmin Yang, Ronald Sederoff & Vincent Chiang ### Abstract Here we describe a protocol for validating expression of artificial microRNAs (amiRNAs) by poly(A) tailing-based RT-PCR. Total RNAs, including amiRNA, are poly(A) tailed using E.coli. poly(A) polymerase. Poly(A) tailed amiRNA can be converted into cDNA along with mRNAs in a reverse transcription reaction primed by a standard poly(T) anchor adaptor. AmiRNA can then be amplified and quantitated by real-tim...

  17. Analysis of several methods for the extraction of high quality DNA from acetic acid bacteria in wine and vinegar for characterization by PCR-based methods. (United States)

    Jara, C; Mateo, E; Guillamón, J M; Torija, M J; Mas, A


    Acetic acid bacteria (AAB) are fastidious microorganisms with poor recovery in culture. Culture-independent methods are currently under examination. Good DNA extraction is a strict requirement of these methods. We compared five methods for extracting the DNA of AAB directly from wine and vinegar samples. Four matrices (white wine, red wine, superficial vinegar and submerged vinegar) contaminated with two AAB strains belonging to Acetobacter pasteurianus and Gluconacetobacter hansenii were assayed. To improve the yield and quality of the extracted DNA, a sample treatment (washing with polyvinyl pyrrolidone or NaCl) was also tested. DNA quality was measured by amplification of the 16S rRNA gene with conventional PCR. DNA recovery rate was assessed by real-time PCR. DNA amplification was always successful with the Wizard method though DNA recovery was poor. A CTAB-based method and NucleoSpin protocol extracted the highest DNA recoveries from wine and vinegar samples. Both of these methods require treatment to recover suitable DNA for amplification with maximum recovery. Both may therefore be good solutions for DNA extraction in wine and vinegar samples. DNA extraction of Ga hansenii was more effective than that of A. pasteurianus. The fastest and cheapest method we evaluated (the Thermal shock protocol) produced the worst results both for DNA amplification and DNA recovery.

  18. Proton-induced single electron capture on DNA/RNA bases. (United States)

    Champion, C; Weck, P F; Lekadir, H; Galassi, M E; Fojón, O A; Abufager, P; Rivarola, R D; Hanssen, J


    In this work, we report total cross sections for the single electron capture process induced on DNA/RNA bases by high-energy protons. The calculations are performed within both the continuum distorted wave and the continuum distorted wave-eikonal initial state approximations. The biological targets are described within the framework of self-consistent methods based on the complete neglect of differential overlap model whose accuracy has first been checked for simpler bio-molecules such as water vapour. Furthermore, the multi-electronic problem investigated here is reduced to a mono-electronic one using a version of the independent electron approximation. Finally, the obtained theoretical predictions are confronted with the scarcely available experimental results.

  19. Post-processing of Deep Web Information Extraction Based on Domain Ontology

    Directory of Open Access Journals (Sweden)

    PENG, T.


    Full Text Available Many methods are utilized to extract and process query results in deep Web, which rely on the different structures of Web pages and various designing modes of databases. However, some semantic meanings and relations are ignored. So, in this paper, we present an approach for post-processing deep Web query results based on domain ontology which can utilize the semantic meanings and relations. A block identification model (BIM based on node similarity is defined to extract data blocks that are relevant to specific domain after reducing noisy nodes. Feature vector of domain books is obtained by result set extraction model (RSEM based on vector space model (VSM. RSEM, in combination with BIM, builds the domain ontology on books which can not only remove the limit of Web page structures when extracting data information, but also make use of semantic meanings of domain ontology. After extracting basic information of Web pages, a ranking algorithm is adopted to offer an ordered list of data records to users. Experimental results show that BIM and RSEM extract data blocks and build domain ontology accurately. In addition, relevant data records and basic information are extracted and ranked. The performances precision and recall show that our proposed method is feasible and efficient.

  20. Theragnosis-based combined cancer therapy using doxorubicin-conjugated microRNA-221 molecular beacon. (United States)

    Lee, Jonghwan; Choi, Kyung-Ju; Moon, Sung Ung; Kim, Soonhag


    Recently, microRNA (miRNA or miR) has emerged as a new cancer biomarker because of its high expression level in various cancer types and its role in the control of tumor suppressor genes. In cancer studies, molecular imaging and treatment based on target cancer markers have been combined to facilitate simultaneous cancer diagnosis and therapy. In this study, for combined therapy with diagnosis of cancer, we developed a doxorubicin-conjugated miR-221 molecular beacon (miR-221 DOXO MB) in a single platform composed of three different nucleotides: miR-221 binding sequence, black hole quencher 1 (BHQ1), and doxorubicin binding site. Imaging of endogenous miR-221 was achieved by specific hybridization between miR-221 and the miR-221 binding site in miR-221 DOXO MB. The presence of miR-221 triggered detachment of the quencher oligo and subsequent activation of a fluorescent signal of miR-221 DOXO MB. Simultaneous cancer therapy in C6 astrocytoma cells and nude mice was achieved by inhibition of miRNA-221 function that downregulates tumor suppressor genes. The detection of miR-221 expression and inhibition of miR-221 function by miR-221 DOXO MB provide the feasibility as a cancer theragnostic probe. Furthermore, a cytotoxic effect was induced by unloading of doxorubicin intercalated into miR-221 DOXO MB inside cells. Loss of miR-221 function and cytotoxicity induced by the miR-221 DOXO MB provides combined therapeutic efficacy against cancers. This method could be used as a new theragnostic probe with enhanced therapy to detect and inhibit many cancer-related miRNAs.

  1. An Efficient Minimum Free Energy Structure-Based Search Method for Riboswitch Identification Based on Inverse RNA Folding. (United States)

    Drory Retwitzer, Matan; Kifer, Ilona; Sengupta, Supratim; Yakhini, Zohar; Barash, Danny


    Riboswitches are RNA genetic control elements that were originally discovered in bacteria and provide a unique mechanism of gene regulation. They work without the participation of proteins and are believed to represent ancient regulatory systems in the evolutionary timescale. One of the biggest challenges in riboswitch research is to find additional eukaryotic riboswitches since more than 20 riboswitch classes have been found in prokaryotes but only one class has been found in eukaryotes. Moreover, this single known class of eukaryotic riboswitch, namely the TPP riboswitch class, has been found in bacteria, archaea, fungi and plants but not in animals. The few examples of eukaryotic riboswitches were identified using sequence-based bioinformatics search methods such as a combination of BLAST and pattern matching techniques that incorporate base-pairing considerations. None of these approaches perform energy minimization structure predictions. There is a clear motivation to develop new bioinformatics methods, aside of the ongoing advances in covariance models, that will sample the sequence search space more flexibly using structural guidance while retaining the computational efficiency of sequence-based methods. We present a new energy minimization approach that transforms structure-based search into a sequence-based search, thereby enabling the utilization of well established sequence-based search utilities such as BLAST and FASTA. The transformation to sequence space is obtained by using an extended inverse RNA folding problem solver with sequence and structure constraints, available within RNAfbinv. Examples in applying the new method are presented for the purine and preQ1 riboswitches. The method is described in detail along with its findings in prokaryotes. Potential uses in finding novel eukaryotic riboswitches and optimizing pre-designed synthetic riboswitches based on ligand simulations are discussed. The method components are freely available for use.

  2. An Efficient Minimum Free Energy Structure-Based Search Method for Riboswitch Identification Based on Inverse RNA Folding.

    Directory of Open Access Journals (Sweden)

    Matan Drory Retwitzer

    Full Text Available Riboswitches are RNA genetic control elements that were originally discovered in bacteria and provide a unique mechanism of gene regulation. They work without the participation of proteins and are believed to represent ancient regulatory systems in the evolutionary timescale. One of the biggest challenges in riboswitch research is to find additional eukaryotic riboswitches since more than 20 riboswitch classes have been found in prokaryotes but only one class has been found in eukaryotes. Moreover, this single known class of eukaryotic riboswitch, namely the TPP riboswitch class, has been found in bacteria, archaea, fungi and plants but not in animals. The few examples of eukaryotic riboswitches were identified using sequence-based bioinformatics search methods such as a combination of BLAST and pattern matching techniques that incorporate base-pairing considerations. None of these approaches perform energy minimization structure predictions. There is a clear motivation to develop new bioinformatics methods, aside of the ongoing advances in covariance models, that will sample the sequence search space more flexibly using structural guidance while retaining the computational efficiency of sequence-based methods. We present a new energy minimization approach that transforms structure-based search into a sequence-based search, thereby enabling the utilization of well established sequence-based search utilities such as BLAST and FASTA. The transformation t