Identifying Stable Reference Genes for qRT-PCR Normalisation in Gene Expression Studies of Narrow-Leafed Lupin (Lupinus angustifolius L..

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Candy M Taylor

Full Text Available Quantitative Reverse Transcription PCR (qRT-PCR is currently one of the most popular, high-throughput and sensitive technologies available for quantifying gene expression. Its accurate application depends heavily upon normalisation of gene-of-interest data with reference genes that are uniformly expressed under experimental conditions. The aim of this study was to provide the first validation of reference genes for Lupinus angustifolius (narrow-leafed lupin, a significant grain legume crop using a selection of seven genes previously trialed as reference genes for the model legume, Medicago truncatula. In a preliminary evaluation, the seven candidate reference genes were assessed on the basis of primer specificity for their respective targeted region, PCR amplification efficiency, and ability to discriminate between cDNA and gDNA. Following this assessment, expression of the three most promising candidates [Ubiquitin C (UBC, Helicase (HEL, and Polypyrimidine tract-binding protein (PTB] was evaluated using the NormFinder and RefFinder statistical algorithms in two narrow-leafed lupin lines, both with and without vernalisation treatment, and across seven organ types (cotyledons, stem, leaves, shoot apical meristem, flowers, pods and roots encompassing three developmental stages. UBC was consistently identified as the most stable candidate and has sufficiently uniform expression that it may be used as a sole reference gene under the experimental conditions tested here. However, as organ type and developmental stage were associated with greater variability in relative expression, it is recommended using UBC and HEL as a pair to achieve optimal normalisation. These results highlight the importance of rigorously assessing candidate reference genes for each species across a diverse range of organs and developmental stages. With emerging technologies, such as RNAseq, and the completion of valuable transcriptome data sets, it is possible that other

Predator-induced defences in Daphnia pulex: Selection and evaluation of internal reference genes for gene expression studies with real-time PCR

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Gilbert Don

2010-06-01

Full Text Available Abstract Background The planktonic microcrustacean Daphnia pulex is among the best-studied animals in ecological, toxicological and evolutionary research. One aspect that has sustained interest in the study system is the ability of D. pulex to develop inducible defence structures when exposed to predators, such as the phantom midge larvae Chaoborus. The available draft genome sequence for D. pulex is accelerating research to identify genes that confer plastic phenotypes that are regularly cued by environmental stimuli. Yet for quantifying gene expression levels, no experimentally validated set of internal control genes exists for the accurate normalization of qRT-PCR data. Results In this study, we tested six candidate reference genes for normalizing transcription levels of D. pulex genes; alpha tubulin (aTub, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, TATA box binding protein (Tbp syntaxin 16 (Stx16, X-box binding protein 1 (Xbp1 and CAPON, a protein associated with the neuronal nitric oxide synthase, were selected on the basis of an earlier study and from microarray studies. One additional gene, a matrix metalloproteinase (MMP, was tested to validate its transcriptional response to Chaoborus, which was earlier observed in a microarray study. The transcription profiles of these seven genes were assessed by qRT-PCR from RNA of juvenile D. pulex that showed induced defences in comparison to untreated control animals. We tested the individual suitability of genes for expression normalization using the programs geNorm, NormFinder and BestKeeper. Intriguingly, Xbp1, Tbp, CAPON and Stx16 were selected as ideal reference genes. Analyses on the relative expression level using the software REST showed that both classical housekeeping candidate genes (aTub and GAPDH were significantly downregulated, whereas the MMP gene was shown to be significantly upregulated, as predicted. aTub is a particularly ill suited reference gene because five copies are

Selection and Validation of Reference Genes for qRT-PCR Expression Analysis of Candidate Genes Involved in Olfactory Communication in the Butterfly Bicyclus anynana

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Arun, Alok; Bauml?, V?ronique; Amelot, Ga?l; Nieberding, Caroline M.

2015-01-01

Real-time quantitative reverse transcription PCR (qRT-PCR) is a technique widely used to quantify the transcriptional expression level of candidate genes. qRT-PCR requires the selection of one or several suitable reference genes, whose expression profiles remain stable across conditions, to normalize the qRT-PCR expression profiles of candidate genes. Although several butterfly species (Lepidoptera) have become important models in molecular evolutionary ecology, so far no study aimed at ident...

A novel mean-centering method for normalizing microRNA expression from high-throughput RT-qPCR data

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Wylie Dennis

2011-12-01

Full Text Available Abstract Background Normalization is critical for accurate gene expression analysis. A significant challenge in the quantitation of gene expression from biofluids samples is the inability to quantify RNA concentration prior to analysis, underscoring the need for robust normalization tools for this sample type. In this investigation, we evaluated various methods of normalization to determine the optimal approach for quantifying microRNA (miRNA expression from biofluids and tissue samples when using the TaqMan® Megaplex™ high-throughput RT-qPCR platform with low RNA inputs. Findings We compared seven normalization methods in the analysis of variation of miRNA expression from biofluid and tissue samples. We developed a novel variant of the common mean-centering normalization strategy, herein referred to as mean-centering restricted (MCR normalization, which is adapted to the TaqMan Megaplex RT-qPCR platform, but is likely applicable to other high-throughput RT-qPCR-based platforms. Our results indicate that MCR normalization performs comparable to or better than both standard mean-centering and other normalization methods. We also propose an extension of this method to be used when migrating biomarker signatures from Megaplex to singleplex RT-qPCR platforms, based on the identification of a small number of normalizer miRNAs that closely track the mean of expressed miRNAs. Conclusions We developed the MCR method for normalizing miRNA expression from biofluids samples when using the TaqMan Megaplex RT-qPCR platform. Our results suggest that normalization based on the mean of all fully observed (fully detected miRNAs minimizes technical variance in normalized expression values, and that a small number of normalizer miRNAs can be selected when migrating from Megaplex to singleplex assays. In our study, we find that normalization methods that focus on a restricted set of miRNAs tend to perform better than methods that focus on all miRNAs, including

Identification of internal control genes for quantitative expression analysis by real-time PCR in bovine peripheral lymphocytes.

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Spalenza, Veronica; Girolami, Flavia; Bevilacqua, Claudia; Riondato, Fulvio; Rasero, Roberto; Nebbia, Carlo; Sacchi, Paola; Martin, Patrice

2011-09-01

Gene expression studies in blood cells, particularly lymphocytes, are useful for monitoring potential exposure to toxicants or environmental pollutants in humans and livestock species. Quantitative PCR is the method of choice for obtaining accurate quantification of mRNA transcripts although variations in the amount of starting material, enzymatic efficiency, and the presence of inhibitors can lead to evaluation errors. As a result, normalization of data is of crucial importance. The most common approach is the use of endogenous reference genes as an internal control, whose expression should ideally not vary among individuals and under different experimental conditions. The accurate selection of reference genes is therefore an important step in interpreting quantitative PCR studies. Since no systematic investigation in bovine lymphocytes has been performed, the aim of the present study was to assess the expression stability of seven candidate reference genes in circulating lymphocytes collected from 15 dairy cows. Following the characterization by flow cytometric analysis of the cell populations obtained from blood through a density gradient procedure, three popular softwares were used to evaluate the gene expression data. The results showed that two genes are sufficient for normalization of quantitative PCR studies in cattle lymphocytes and that YWAHZ, S24 and PPIA are the most stable genes. Copyright © 2010 Elsevier Ltd. All rights reserved.

Quantitative Real Time PCR approach to study gene expression profile during prenatal growth of skeletal muscle in pig of Duroc and Pietrain breeds

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M. Cagnazzo

2010-01-01

Full Text Available The quantitative real time-PCR (QRT-PCR is a very sensitive method used to quantify mRNA level in gene expression analysis. Combining amplification, detection and quantification in a single step, allows a more accurate measurement compared to the traditional PCR end point analysis (Pfaffl, 2001; Bustin, 2002.

PCR Expression Analysis Of the Estrogeninducible Gene Bcei in Gastrointestinal and Other Human Tumors

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Iris Wundrack

1994-01-01

Full Text Available A polymerase chain reaction (PCR assay was developed to test for tumor cell specific expression of the BCEI gene. This new marker gene, reported at first for human breast cancer, was found specifically active in various gastrointestinal carcinomas by previously applying immunohistochemistry and RNA (Northern blot analysis. Presently, by using reverse transcription -PCR analysis, a series of primary tumor tissues and established tumor cell lines were testcd for BCEI transcription. This approach was compared to immunostaining achieved by an antibody directed against the BCEI gene’s product. The result demonstrate the superior sensitivity of PCR by indicating the gene’ s expression in cases where immunohistochemical testing remained negative.

Complementary techniques: validation of gene expression data by quantitative real time PCR.

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Provenzano, Maurizio; Mocellin, Simone

2007-01-01

Microarray technology can be considered the most powerful tool for screening gene expression profiles of biological samples. After data mining, results need to be validated with highly reliable biotechniques allowing for precise quantitation of transcriptional abundance of identified genes. Quantitative real time PCR (qrt-PCR) technology has recently reached a level of sensitivity, accuracy and practical ease that support its use as a routine bioinstrumentation for gene level measurement. Currently, qrt-PCR is considered by most experts the most appropriate method to confirm or confute microarray-generated data. The knowledge of the biochemical principles underlying qrt-PCR as well as some related technical issues must be beard in mind when using this biotechnology.

Normalizing gene expression by quantitative PCR during somatic embryogenesis in two representative conifer species: Pinus pinaster and Picea abies.

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de Vega-Bartol, José J; Santos, Raquen Raissa; Simões, Marta; Miguel, Célia M

2013-05-01

Suitable internal control genes to normalize qPCR data from different stages of embryo development and germination were identified in two representative conifer species. Clonal propagation by somatic embryogenesis has a great application potentiality in conifers. Quantitative PCR (qPCR) is widely used for gene expression analysis during somatic embryogenesis and embryo germination. No single reference gene is universal, so a systematic characterization of endogenous genes for concrete conditions is fundamental for accuracy. We identified suitable internal control genes to normalize qPCR data obtained at different steps of somatic embryogenesis (embryonal mass proliferation, embryo maturation and germination) in two representative conifer species, Pinus pinaster and Picea abies. Candidate genes included endogenous genes commonly used in conifers, genes previously tested in model plants, and genes with a lower variation of the expression along embryo development according to genome-wide transcript profiling studies. Three different algorithms were used to evaluate expression stability. The geometric average of the expression values of elongation factor-1α, α-tubulin and histone 3 in P. pinaster, and elongation factor-1α, α-tubulin, adenosine kinase and CAC in P. abies were adequate for expression studies throughout somatic embryogenesis. However, improved accuracy was achieved when using other gene combinations in experiments with samples at a single developmental stage. The importance of studies selecting reference genes to use in different tissues or developmental stages within one or close species, and the instability of commonly used reference genes, is highlighted.

A method for quantitative analysis of standard and high-throughput qPCR expression data based on input sample quantity.

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Mateusz G Adamski

Full Text Available Over the past decade rapid advances have occurred in the understanding of RNA expression and its regulation. Quantitative polymerase chain reactions (qPCR have become the gold standard for quantifying gene expression. Microfluidic next generation, high throughput qPCR now permits the detection of transcript copy number in thousands of reactions simultaneously, dramatically increasing the sensitivity over standard qPCR. Here we present a gene expression analysis method applicable to both standard polymerase chain reactions (qPCR and high throughput qPCR. This technique is adjusted to the input sample quantity (e.g., the number of cells and is independent of control gene expression. It is efficiency-corrected and with the use of a universal reference sample (commercial complementary DNA (cDNA permits the normalization of results between different batches and between different instruments--regardless of potential differences in transcript amplification efficiency. Modifications of the input quantity method include (1 the achievement of absolute quantification and (2 a non-efficiency corrected analysis. When compared to other commonly used algorithms the input quantity method proved to be valid. This method is of particular value for clinical studies of whole blood and circulating leukocytes where cell counts are readily available.

In situ PCR detection and significance of IL-3 gene expression in irradiated hematopoietic cells of mouse bone marrow

International Nuclear Information System (INIS)

Peng Ruiyun; Wang Dewen; Xiong Chengqi; Gao Yabing; Li Yanping; Yang Hong; Cui Yufang

2000-01-01

Objective: To study the significance of endogenous interleukin 3(IL-3) gene expression in repair of irradiated mouse bone marrow. Methods: Seventy-eight LACA mice were subjected to total body irradiation with 60 Co γ-rays and were sacrificed within 4 weeks after irradiation. The bone marrow histopathological sections were stained with HE, and the expression of endogenous IL-3 gene was detected by means of immunocytochemistry,in situ hybridization(ISH) and in situ reverse transcription PCR(IS RT-PCR). Results: Obvious injury of bone marrow occurred after irradiation and then recovered within 4 weeks. IL-3 protein was obviously increased in the cytoplasm of recovering hematopoietic cells(HCs), especially on day 21 after irradiation, while its mRNA was poorly positive by ISH on days 10-21, especially day 15.IS RT-PCR showed that IL-3 mRNA was strongly positive in recovering HCs cytoplasm, especially on days 10 to 15. Conclusion: In situ RT-PCR can objectively reflect the regulation of IL-3 gene expression in bone marrow after irradiation, and the expression of endogenous IL-3 gene may play an important role in hematopoietic reconstruction of irradiated bone marrow

Selection and evaluation of reference genes for expression studies with quantitative PCR in the model fungus Neurospora crassa under different environmental conditions in continuous culture.

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Kathleen D Cusick

Full Text Available Neurospora crassa has served as a model organism for studying circadian pathways and more recently has gained attention in the biofuel industry due to its enhanced capacity for cellulase production. However, in order to optimize N. crassa for biotechnological applications, metabolic pathways during growth under different environmental conditions must be addressed. Reverse-transcription quantitative PCR (RT-qPCR is a technique that provides a high-throughput platform from which to measure the expression of a large set of genes over time. The selection of a suitable reference gene is critical for gene expression studies using relative quantification, as this strategy is based on normalization of target gene expression to a reference gene whose expression is stable under the experimental conditions. This study evaluated twelve candidate reference genes for use with N. crassa when grown in continuous culture bioreactors under different light and temperature conditions. Based on combined stability values from NormFinder and Best Keeper software packages, the following are the most appropriate reference genes under conditions of: (1 light/dark cycling: btl, asl, and vma1; (2 all-dark growth: btl, tbp, vma1, and vma2; (3 temperature flux: btl, vma1, act, and asl; (4 all conditions combined: vma1, vma2, tbp, and btl. Since N. crassa exists as different cell types (uni- or multi-nucleated, expression changes in a subset of the candidate genes was further assessed using absolute quantification. A strong negative correlation was found to exist between ratio and threshold cycle (CT values, demonstrating that CT changes serve as a reliable reflection of transcript, and not gene copy number, fluctuations. The results of this study identified genes that are appropriate for use as reference genes in RT-qPCR studies with N. crassa and demonstrated that even with the presence of different cell types, relative quantification is an acceptable method for measuring

Selection and evaluation of reference genes for expression studies with quantitative PCR in the model fungus Neurospora crassa under different environmental conditions in continuous culture.

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Cusick, Kathleen D; Fitzgerald, Lisa A; Pirlo, Russell K; Cockrell, Allison L; Petersen, Emily R; Biffinger, Justin C

2014-01-01

Neurospora crassa has served as a model organism for studying circadian pathways and more recently has gained attention in the biofuel industry due to its enhanced capacity for cellulase production. However, in order to optimize N. crassa for biotechnological applications, metabolic pathways during growth under different environmental conditions must be addressed. Reverse-transcription quantitative PCR (RT-qPCR) is a technique that provides a high-throughput platform from which to measure the expression of a large set of genes over time. The selection of a suitable reference gene is critical for gene expression studies using relative quantification, as this strategy is based on normalization of target gene expression to a reference gene whose expression is stable under the experimental conditions. This study evaluated twelve candidate reference genes for use with N. crassa when grown in continuous culture bioreactors under different light and temperature conditions. Based on combined stability values from NormFinder and Best Keeper software packages, the following are the most appropriate reference genes under conditions of: (1) light/dark cycling: btl, asl, and vma1; (2) all-dark growth: btl, tbp, vma1, and vma2; (3) temperature flux: btl, vma1, act, and asl; (4) all conditions combined: vma1, vma2, tbp, and btl. Since N. crassa exists as different cell types (uni- or multi-nucleated), expression changes in a subset of the candidate genes was further assessed using absolute quantification. A strong negative correlation was found to exist between ratio and threshold cycle (CT) values, demonstrating that CT changes serve as a reliable reflection of transcript, and not gene copy number, fluctuations. The results of this study identified genes that are appropriate for use as reference genes in RT-qPCR studies with N. crassa and demonstrated that even with the presence of different cell types, relative quantification is an acceptable method for measuring gene

Identification and validation of quantitative real-time reverse transcription PCR reference genes for gene expression analysis in teak (Tectona grandis L.f.).

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Galeano, Esteban; Vasconcelos, Tarcísio Sales; Ramiro, Daniel Alves; De Martin, Valentina de Fátima; Carrer, Helaine

2014-07-22

Teak (Tectona grandis L.f.) is currently the preferred choice of the timber trade for fabrication of woody products due to its extraordinary qualities and is widely grown around the world. Gene expression studies are essential to explore wood formation of vascular plants, and quantitative real-time reverse transcription PCR (qRT-PCR) is a sensitive technique employed for quantifying gene expression levels. One or more appropriate reference genes are crucial to accurately compare mRNA transcripts through different tissues/organs and experimental conditions. Despite being the focus of some genetic studies, a lack of molecular information has hindered genetic exploration of teak. To date, qRT-PCR reference genes have not been identified and validated for teak. Identification and cloning of nine commonly used qRT-PCR reference genes from teak, including ribosomal protein 60s (rp60s), clathrin adaptor complexes medium subunit family (Cac), actin (Act), histone 3 (His3), sand family (Sand), β-Tubulin (Β-Tub), ubiquitin (Ubq), elongation factor 1-α (Ef-1α), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Expression profiles of these genes were evaluated by qRT-PCR in six tissue and organ samples (leaf, flower, seedling, root, stem and branch secondary xylem) of teak. Appropriate gene cloning and sequencing, primer specificity and amplification efficiency was verified for each gene. Their stability as reference genes was validated by NormFinder, BestKeeper, geNorm and Delta Ct programs. Results obtained from all programs showed that TgUbq and TgEf-1α are the most stable genes to use as qRT-PCR reference genes and TgAct is the most unstable gene in teak. The relative expression of the teak cinnamyl alcohol dehydrogenase (TgCAD) gene in lignified tissues at different ages was assessed by qRT-PCR, using TgUbq and TgEf-1α as internal controls. These analyses exposed a consistent expression pattern with both reference genes. This study proposes a first broad

Correlation Coefficients Between Different Methods of Expressing Bacterial Quantification Using Real Time PCR

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Bahman Navidshad

2012-02-01

Full Text Available The applications of conventional culture-dependent assays to quantify bacteria populations are limited by their dependence on the inconsistent success of the different culture-steps involved. In addition, some bacteria can be pathogenic or a source of endotoxins and pose a health risk to the researchers. Bacterial quantification based on the real-time PCR method can overcome the above-mentioned problems. However, the quantification of bacteria using this approach is commonly expressed as absolute quantities even though the composition of samples (like those of digesta can vary widely; thus, the final results may be affected if the samples are not properly homogenized, especially when multiple samples are to be pooled together before DNA extraction. The objective of this study was to determine the correlation coefficients between four different methods of expressing the output data of real-time PCR-based bacterial quantification. The four methods were: (i the common absolute method expressed as the cell number of specific bacteria per gram of digesta; (ii the Livak and Schmittgen, ΔΔCt method; (iii the Pfaffl equation; and (iv a simple relative method based on the ratio of cell number of specific bacteria to the total bacterial cells. Because of the effect on total bacteria population in the results obtained using ΔCt-based methods (ΔΔCt and Pfaffl, these methods lack the acceptable consistency to be used as valid and reliable methods in real-time PCR-based bacterial quantification studies. On the other hand, because of the variable compositions of digesta samples, a simple ratio of cell number of specific bacteria to the corresponding total bacterial cells of the same sample can be a more accurate method to quantify the population.

Selection of housekeeping genes for normalization by real-time RT-PCR: analysis of Or-MYB1 gene expression in Orobanche ramosa development.

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González-Verdejo, C I; Die, J V; Nadal, S; Jiménez-Marín, A; Moreno, M T; Román, B

2008-08-15

Real-time PCR has become the method of choice for accurate and in-depth expression studies of candidate genes. To avoid bias, real-time PCR is referred to one or several internal control genes that should not fluctuate among treatments. A need for reference genes in the parasitic plant Orobanche ramosa has emerged, and the studies in this area have not yet been evaluated. In this study, the genes 18S rRNA, Or-act1, Or-tub1, and Or-ubq1 were compared in terms of expression stability using the BestKeeper software program. Among the four common endogenous control genes, Or-act1 and Or-ubq1 were the most stable in O. ramosa samples. In parallel, a study was carried out studying the expression of the transcription factor Or-MYB1 that seemed to be implicated during preinfection stages. The normalization strategy presented here is a prerequisite to accurate real-time PCR expression profiling that, among other things, opens up the possibility of studying messenger RNA levels of low-copy-number-like transcription factors.

Quantitative RT-PCR analysis of estrogen receptor gene expression in laser microdissected prostate cancer tissue.

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Walton, Thomas J; Li, Geng; McCulloch, Thomas A; Seth, Rashmi; Powe, Desmond G; Bishop, Michael C; Rees, Robert C

2009-06-01

Real-time quantitative RT-PCR analysis of laser microdissected tissue is considered the most accurate technique for determining tissue gene expression. The discovery of estrogen receptor beta (ERbeta) has focussed renewed interest on the role of estrogen receptors in prostate cancer, yet few studies have utilized the technique to analyze estrogen receptor gene expression in prostate cancer. Fresh tissue was obtained from 11 radical prostatectomy specimens and from 6 patients with benign prostate hyperplasia. Pure populations of benign and malignant prostate epithelium were laser microdissected, followed by RNA isolation and electrophoresis. Quantitative RT-PCR was performed using primers for androgen receptor (AR), estrogen receptor beta (ERbeta), estrogen receptor alpha (ERalpha), progesterone receptor (PGR) and prostate specific antigen (PSA), with normalization to two housekeeping genes. Differences in gene expression were analyzed using the Mann-Whitney U-test. Correlation coefficients were analyzed using Spearman's test. Significant positive correlations were seen when AR and AR-dependent PSA, and ERalpha and ERalpha-dependent PGR were compared, indicating a representative population of RNA transcripts. ERbeta gene expression was significantly over-expressed in the cancer group compared with benign controls (P cancer group (P prostate cancer specimens. In concert with recent studies the findings suggest differential production of ERbeta splice variants, which may play important roles in the genesis of prostate cancer. (c) 2009 Wiley-Liss, Inc.

Merkel cell polyomavirus detection in Merkel cell cancer tumors in Northern Germany using PCR and protein expression.

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Leitz, Miriam; Stieler, Kristin; Grundhoff, Adam; Moll, Ingrid; Brandner, Johanna M; Fischer, Nicole

2014-10-01

Merkel cell carcinoma is a highly malignant skin cancer which predominantly occurs in elderly and immunocompromised persons. The identification of the Merkel cell polyomavirus (MCPyV) has inaugurated a new understanding of Merkel cell carcinoma pathogenesis. The frequent detection of the virus in Merkel cell carcinoma tissue (70-90%), its monoclonal integration in the tumor cells and the expression of viral oncogenes highly suggest that MCPyV is causally linked to the pathogenesis of the majority of Merkel cell cancer (MCC) cases. Using qualitative and quantitative PCR together with immunohistochemical staining this study aimed at characterizing the presence of MCPyV sequences and viral early gene expression in a cohort of MCC cases (n = 32) selected in Northern Germany. 40-57% of the cases were identified as MCPyV positive with 40.6% of the cases positive by immunohistochemical staining and 51.6-57.6% positive by PCR. Interestingly, in the majority (64%) of LT-Antigen positive tumors only 25-50% of tumor cells express LT-Antigen. These data are in accord with published studies describing heterogeneity in MCPyV viral loads and suggest that detection of MCPyV in Merkel cell carcinoma by PCR should be undertaken using multiple primer pairs. © 2013 Wiley Periodicals, Inc.

Identification of reference genes for expression analysis by real-time quantitative PCR in drought-stressed soybean

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Renata Stolf-Moreira

2011-01-01

Full Text Available The objective of this work was to validate, by quantitative PCR in real time (RT-qPCR, genes to be used as reference in studies of gene expression in soybean in drought-stressed trials. Four genes commonly used in soybean were evaluated: Gmβ-actin, GmGAPDH, GmLectin and GmRNAr18S. Total RNA was extracted from six samples: three from roots in a hydroponic system with different drought intensities (0, 25, 50, 75 and 100 minutes of water stress, and three from leaves of plants grown in sand with different soil moistures (15, 5 and 2.5% gravimetric humidity. The raw cycle threshold (Ct data were analyzed, and the efficiency of each primer was calculated for an overall analysis of the Ct range among the different samples. The GeNorm application was used to evaluate the best reference gene, according to its stability. The GmGAPDH was the least stable gene, with the highest mean values of expression stability (M, and the most stable genes, with the lowest M values, were the Gmβ-actin and GmRNAr18S, when both root and leaves samples were tested. These genes can be used in RT-qPCR as reference gene for expression analysis.

Analysis on the arcelin expression in bruchid pest resistant wild pulses using real time RT-qPCR.

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Sakthivelkumar, Shanmugavel; Veeramani, Velayutham; Hilda, Karuppiah; Arumugam, Munusamy; Janarthanan, Sundaram

2014-12-01

Arcelin, the antimetabolic protein from wild pulses is a known natural insecticidal molecule. Wild pulses with high arcelin content could serve as potential source to. increase the levels of insect resistance in cultivated pulse crops. In this study, arcelin (Arl) gene expression was screened in seven stored product insect pest resistant wild pulse varieties using real time RT-qPCR. Arcelin gene specific real time PCR primers were synthesized from arcelin mRNA sequence of the wild pulse variety, Lablab purpureus. The results revealed different levels of arcelin gene expression in the tested varieties. Canavalia virosa registered significantly high content indicating its suitability for utilization of arcelin gene in developing stored product insect pest resistance with other cultivated pulses.

Assessment of brain reference genes for RT-qPCR studies in neurodegenerative diseases.

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Rydbirk, Rasmus; Folke, Jonas; Winge, Kristian; Aznar, Susana; Pakkenberg, Bente; Brudek, Tomasz

2016-11-17

Evaluation of gene expression levels by reverse transcription quantitative real-time PCR (RT-qPCR) has for many years been the favourite approach for discovering disease-associated alterations. Normalization of results to stably expressed reference genes (RGs) is pivotal to obtain reliable results. This is especially important in relation to neurodegenerative diseases where disease-related structural changes may affect the most commonly used RGs. We analysed 15 candidate RGs in 98 brain samples from two brain regions from Alzheimer's disease (AD), Parkinson's disease (PD), Multiple System Atrophy, and Progressive Supranuclear Palsy patients. Using RefFinder, a web-based tool for evaluating RG stability, we identified the most stable RGs to be UBE2D2, CYC1, and RPL13 which we recommend for future RT-qPCR studies on human brain tissue from these patients. None of the investigated genes were affected by experimental variables such as RIN, PMI, or age. Findings were further validated by expression analyses of a target gene GSK3B, known to be affected by AD and PD. We obtained high variations in GSK3B levels when contrasting the results using different sets of common RG underlining the importance of a priori validation of RGs for RT-qPCR studies.

Characterizing bacterial gene expression in nitrogen cycle metabolism with RT-qPCR.

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Graham, James E; Wantland, Nicholas B; Campbell, Mark; Klotz, Martin G

2011-01-01

Recent advances in DNA sequencing have greatly accelerated our ability to obtain the raw information needed to recognize both known and potential novel modular microbial genomic capacity for nitrogen metabolism. With PCR-based approaches to quantifying microbial mRNA expression now mainstream in most laboratories, researchers can now more efficiently propose and test hypotheses on the contributions of individual microbes to the biological accessibility of nitrogen upon which all other life depends. We review known microbial roles in these key nitrogen transformations, and describe the necessary steps in carrying out relevant gene expression studies. An example experimental design is then provided characterizing Nitrosococcus oceani mRNA expression in cultures responding to ammonia. The approach described, that of assessing microbial genome inventory and testing putative modular gene expression by mRNA quantification, is likely to remain an important tool in understanding individual microbial contributions within microbial community activities that maintain the Earth's nitrogen balance. Copyright © 2011 Elsevier Inc. All rights reserved.

Selection and validation of reference genes for gene expression analysis in switchgrass (Panicum virgatum using quantitative real-time RT-PCR.

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Jacinta Gimeno

Full Text Available Switchgrass (Panicum virgatum has received a lot of attention as a forage and bioenergy crop during the past few years. Gene expression studies are in progress to improve new traits and develop new cultivars. Quantitative real time PCR (qRT-PCR has emerged as an important technique to study gene expression analysis. For accurate and reliable results, normalization of data with reference genes is essential. In this work, we evaluate the stability of expression of genes to use as reference for qRT-PCR in the grass P. virgatum. Eleven candidate reference genes, including eEF-1α, UBQ6, ACT12, TUB6, eIF-4a, GAPDH, SAMDC, TUA6, CYP5, U2AF, and FTSH4, were validated for qRT-PCR normalization in different plant tissues and under different stress conditions. The expression stability of these genes was verified by the use of two distinct algorithms, geNorm and NormFinder. Differences were observed after comparison of the ranking of the candidate reference genes identified by both programs but eEF-1α, eIF-4a, CYP5 and U2AF are ranked as the most stable genes in the samples sets under study. Both programs discard the use of SAMDC and TUA6 for normalization. Validation of the reference genes proposed by geNorm and NormFinder were performed by normalization of transcript abundance of a group of target genes in different samples. Results show similar expression patterns when the best reference genes selected by both programs were used but differences were detected in the transcript abundance of the target genes. Based on the above research, we recommend the use of different statistical algorithms to identify the best reference genes for expression data normalization. The best genes selected in this study will help to improve the quality of gene expression data in a wide variety of samples in switchgrass.

A comparative study of three different gene expression analysis methods.

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Choe, Jae Young; Han, Hyung Soo; Lee, Seon Duk; Lee, Hanna; Lee, Dong Eun; Ahn, Jae Yun; Ryoo, Hyun Wook; Seo, Kang Suk; Kim, Jong Kun

2017-12-04

TNF-α regulates immune cells and acts as an endogenous pyrogen. Reverse transcription polymerase chain reaction (RT-PCR) is one of the most commonly used methods for gene expression analysis. Among the alternatives to PCR, loop-mediated isothermal amplification (LAMP) shows good potential in terms of specificity and sensitivity. However, few studies have compared RT-PCR and LAMP for human gene expression analysis. Therefore, in the present study, we compared one-step RT-PCR, two-step RT-LAMP and one-step RT-LAMP for human gene expression analysis. We compared three gene expression analysis methods using the human TNF-α gene as a biomarker from peripheral blood cells. Total RNA from the three selected febrile patients were subjected to the three different methods of gene expression analysis. In the comparison of three gene expression analysis methods, the detection limit of both one-step RT-PCR and one-step RT-LAMP were the same, while that of two-step RT-LAMP was inferior. One-step RT-LAMP takes less time, and the experimental result is easy to determine. One-step RT-LAMP is a potentially useful and complementary tool that is fast and reasonably sensitive. In addition, one-step RT-LAMP could be useful in environments lacking specialized equipment or expertise.

Assessment of brain reference genes for RT-qPCR studies in neurodegenerative diseases

DEFF Research Database (Denmark)

Rydbirk, Rasmus; Folke, Jonas; Winge, Kristian

2016-01-01

Evaluation of gene expression levels by reverse transcription quantitative real-time PCR (RT-qPCR) has for many years been the favourite approach for discovering disease-associated alterations. Normalization of results to stably expressed reference genes (RGs) is pivotal to obtain reliable results......, and Progressive Supranuclear Palsy patients. Using RefFinder, a web-based tool for evaluating RG stability, we identified the most stable RGs to be UBE2D2, CYC1, and RPL13 which we recommend for future RT-qPCR studies on human brain tissue from these patients. None of the investigated genes were affected...... by experimental variables such as RIN, PMI, or age. Findings were further validated by expression analyses of a target gene GSK3B, known to be affected by AD and PD. We obtained high variations in GSK3B levels when contrasting the results using different sets of common RG underlining the importance of a priori...

Selection of reference genes for quantitative RT-PCR studies in striped dolphin (Stenella coeruleoalba skin biopsies

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Casini Silvia

2006-09-01

Full Text Available Abstract Background Odontocete cetaceans occupy the top position of the marine food-web and are particularly sensitive to the bioaccumulation of lipophilic contaminants. The effects of environmental pollution on these species are highly debated and various ecotoxicological studies have addressed the impact of xenobiotic compounds on marine mammals, raising conservational concerns. Despite its sensitivity, quantitative real-time PCR (qRT-PCR has never been used to quantify gene induction caused by exposure of cetaceans to contaminants. A limitation for the application of qRT-PCR is the need for appropriate reference genes which allow the correct quantification of gene expression. A systematic evaluation of potential reference genes in cetacean skin biopsies is presented, in order to validate future qRT-PCR studies aiming at using the expression of selected genes as non-lethal biomarkers. Results Ten commonly used housekeeping genes (HKGs were partially sequenced in the striped dolphin (Stenella coeruleoalba and, for each gene, PCR primer pairs were specifically designed and tested in qRT-PCR assays. The expression of these potential control genes was examined in 30 striped dolphin skin biopsy samples, obtained from specimens sampled in the north-western Mediterranean Sea. The stability of selected control genes was determined using three different specific VBA applets (geNorm, NormFinder and BestKeeper which produce highly comparable results. Glyceraldehyde-3P-dehydrogenase (GAPDH and tyrosine 3-monooxygenase (YWHAZ always rank as the two most stably expressed HKGs according to the analysis with geNorm and Normfinder, and are defined as optimal control genes by BestKepeer. Ribosomal protein L4 (RPL4 and S18 (RPS18 also exhibit a remarkable stability of their expression levels. On the other hand, transferrin receptor (TFRC, phosphoglycerate kinase 1 (PGK1, hypoxanthine ribosyltransferase (HPRT1 and β-2-microglobin (B2M show variable expression

Expression Profile of IL-35 mRNA in Gingiva of Chronic Periodontitis and Aggressive Periodontitis Patients: A Semiquantitative RT-PCR Study

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Nagaraj B. Kalburgi

2013-01-01

Full Text Available Background. Proinflammatory and anti-inflammatory cytokines play a key role in the pathogenesis of periodontal diseases. Secretion of bioactive IL-35 has been described by T regulatory cells ( and is required for their maximal suppressive activity. are involved in the modulation of local immune response in chronic periodontitis patients. Objective. Hence, the present study was aimed to investigate the expression of IL-35 mRNA in chronic periodontitis and aggressive periodontitis patients. Materials and Methods. The present study was carried out in 60 subjects, which included 20 chronic periodontitis patients, 20 aggressive periodontitis patients, and 20 periodontally healthy controls. IL-35 mRNA expression in gingival tissue samples of all subjects was semiquantitatively analyzed using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR. Results. The present study demonstrated the expression of IL-35 mRNA in gingival tissues of all the three groups. IL-35 mRNA expression was highest in chronic periodontitis subjects ( as compared to the aggressive periodontitis group ( and least seen in healthy patients (. Conclusion. The increased expression of IL-35 in chronic and aggressive periodontitis suggests its possible role in pathogenesis of periodontitis. Future studies done on large samples with intervention will strengthen our result.

Application of reverse transcription-PCR and real-time PCR in nanotoxicity research.

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Mo, Yiqun; Wan, Rong; Zhang, Qunwei

2012-01-01

Reverse transcription-polymerase chain reaction (RT-PCR) is a relatively simple and inexpensive technique to determine the expression level of target genes and is widely used in biomedical science research including nanotoxicology studies for semiquantitative analysis. Real-time PCR allows for the detection of PCR amplification in the exponential growth phase of the reaction and is much more quantitative than traditional RT-PCR. Although a number of kits and reagents for RT-PCR and real-time PCR are commercially available, the basic principles are the same. Here, we describe the procedures for total RNA isolation by using TRI Reagent, for reverse transcription (RT) by M-MLV reverse transcriptase, and for PCR by GoTaq(®) DNA Polymerase. And real-time PCR will be performed on an iQ5 multicolor real-time PCR detection system by using iQ™ SYBR Green Supermix.

Expression stabilities of candidate reference genes for RT-qPCR under different stress conditions in soybean.

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Shuhua Ma

Full Text Available Due to its accuracy, sensitivity and high throughput, real time quantitative PCR (RT-qPCR has been widely used in analysing gene expression. The quality of data from such analyses is affected by the quality of reference genes used. Expression stabilities for nine candidate reference genes widely used in soybean were evaluated under different stresses in this study. Our results showed that EF1A and ACT11 were the best under salinity stress, TUB4, TUA5 and EF1A were the best under drought stress, ACT11 and UKN2 were the best under dark treatment, and EF1B and UKN2 were the best under virus infection. EF1B and UKN2 were the top two genes which can be reliably used in all of the stress conditions assessed.

Evaluation of reference genes for gene expression analysis using quantitative RT-PCR in Azospirillum brasilense.

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McMillan, Mary; Pereg, Lily

2014-01-01

Azospirillum brasilense is a nitrogen fixing bacterium that has been shown to have various beneficial effects on plant growth and yield. Under normal conditions A. brasilense exists in a motile flagellated form, which, under starvation or stress conditions, can undergo differentiation into an encapsulated, cyst-like form. Quantitative RT-PCR can be used to analyse changes in gene expression during this differentiation process. The accuracy of quantification of mRNA levels by qRT-PCR relies on the normalisation of data against stably expressed reference genes. No suitable set of reference genes has yet been described for A. brasilense. Here we evaluated the expression of ten candidate reference genes (16S rRNA, gapB, glyA, gyrA, proC, pykA, recA, recF, rpoD, and tpiA) in wild-type and mutant A. brasilense strains under different culture conditions, including conditions that induce differentiation. Analysis with the software programs BestKeeper, NormFinder and GeNorm indicated that gyrA, glyA and recA are the most stably expressed reference genes in A. brasilense. The results also suggested that the use of two reference genes (gyrA and glyA) is sufficient for effective normalisation of qRT-PCR data.

Evaluation of reference genes for gene expression analysis using quantitative RT-PCR in Azospirillum brasilense.

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Mary McMillan

Full Text Available Azospirillum brasilense is a nitrogen fixing bacterium that has been shown to have various beneficial effects on plant growth and yield. Under normal conditions A. brasilense exists in a motile flagellated form, which, under starvation or stress conditions, can undergo differentiation into an encapsulated, cyst-like form. Quantitative RT-PCR can be used to analyse changes in gene expression during this differentiation process. The accuracy of quantification of mRNA levels by qRT-PCR relies on the normalisation of data against stably expressed reference genes. No suitable set of reference genes has yet been described for A. brasilense. Here we evaluated the expression of ten candidate reference genes (16S rRNA, gapB, glyA, gyrA, proC, pykA, recA, recF, rpoD, and tpiA in wild-type and mutant A. brasilense strains under different culture conditions, including conditions that induce differentiation. Analysis with the software programs BestKeeper, NormFinder and GeNorm indicated that gyrA, glyA and recA are the most stably expressed reference genes in A. brasilense. The results also suggested that the use of two reference genes (gyrA and glyA is sufficient for effective normalisation of qRT-PCR data.

Validation of RNAi by real time PCR

DEFF Research Database (Denmark)

Josefsen, Knud; Lee, Ying Chiu

2011-01-01

Real time PCR is the analytic tool of choice for quantification of gene expression, while RNAi is concerned with downregulation of gene expression. Together, they constitute a powerful approach in any loss of function studies of selective genes. We illustrate here the use of real time PCR to verify...

Simultaneous Expression of GUS and Actin Genes by Using the Multiplex RT-PCR and Multiplex Gold Nanoparticle Probes.

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Ghazi, Yaser; Vaseghi, Akbar; Ahmadi, Sepideh; Haddadi, Fatemeh

2018-04-23

Gene expression analysis is considered to be extremely important in many different biological researches. DNA-based diagnostic test, which contributes to DNA identification, has higher specificity, cost, and speed than some biochemical and molecular methods. In this study, we try to use the novel nano technology approach with Multiplex RT-PCR and Gold nano particular probes (GNPs-probes) in order to get gene expression in Curcumas melons. We used Agrobacterium tumefactions for gene transfer and GUS reporter gene as a reporter. After cDNA synthesis, Multiplex PCR and Multiplex RT-PCR techniques were used. Finally, probes were designed for RNA of GUS and Actin genes, and then the analysis of the gene expression using the probes attached to GNPs was carried out and the color changes in the GNPs were applied. In the following, probes hybridization was checked with DNA between 400 to 700 nm wavelengths and the highest rate was observed in the 550 to 650 nm. The results show that the simultaneous use of GNP-attached detectors and Multiplex RT-PCRcan reduce time and costmore considerably than somelaboratory methods for gene expiration investigation. Additionally, it can be seen thatthere is an increase in sensitivity and specificity of our investigation. Based on our findings, this can bea novel study doneusingMultiplex RT-PCRand unmodified AuNPs for gene transfer and expression detection to plants. We can claim that this assay has a remarkable advantage including rapid, cost-effectiveness, specificity and accuracy to detect transfer and expression genes in plants. Also,we can use this technique from other gene expressionsin many different biology samples.

Real-Time PCR (qPCR) Primer Design Using Free Online Software

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Thornton, Brenda; Basu, Chhandak

2011-01-01

Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most…

The utility of optical detection system (qPCR) and bioinformatics methods in reference gene expression analysis

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Skarzyńska, Agnieszka; Pawełkowicz, Magdalena; PlÄ der, Wojciech; Przybecki, Zbigniew

2016-09-01

Real-time quantitative polymerase chain reaction is consider as the most reliable method for gene expression studies. However, the expression of target gene could be misinterpreted due to improper normalization. Therefore, the crucial step for analysing of qPCR data is selection of suitable reference genes, which should be validated experimentally. In order to choice the gene with stable expression in the designed experiment, we performed reference gene expression analysis. In this study genes described in the literature and novel genes predicted as control genes, based on the in silico analysis of transcriptome data were used. Analysis with geNorm and NormFinder algorithms allow to create the ranking of candidate genes and indicate the best reference for flower morphogenesis study. According to the results, genes CACS and CYCL were characterised the most stable expression, but the least suitable genes were TUA and EF.

Identification of Suitable Endogenous Normalizers for qRT- PCR Analysis of Plasma microRNA Expression in Essential Hypertension

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Solayman, Mohamed Hassan M.; Langaee, Taimour; Patel, Archanakumari; El-Wakeel, Lamia; El-Hamamsy, Manal; Badary, Osama; Johnson, Julie A.

2016-01-01

Circulating microRNAs (miRNAs) are promising biomarkers for many diseases. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a gold standard for miRNA expression profiling that requires proper data normalization. Since there is no universal normalizer, it is recommended to evaluate normalizers under every experimental condition. This study describes the identification of suitable endogenous normalizer(s) (ENs) for plasma miRNA expression in essential hypertension. Expression levels of 5 candidate ENs and 2 plasma quality markers were determined by qRT-PCR in plasma samples from 18 hypertensive patients and 10 healthy controls. NormFinder, GeNorm, and DataAssist software programs were used to select the best EN(s). Expression levels of the 5 candidate ENs were also analyzed in urine samples from hypertensive patients and compared to the plasma samples of the hypertensive patients. Among the analyzed candidates, hsa-miR-92a-3p was identified as the best EN, and hsa-miR-21-5p and hsa-miR-16-5p as next best. Moreover, hsa-miR-92a-3p showed the most consistent expression between plasma and urine In conclusion, this study showed that hsa-miR-92a-3p, hsa-miR-21-5p, and hsa-miR-16-5p may be used as normalizers for plasma miRNA expression data in essential hypertension studies. PMID:26798072

Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process

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Borges-Pérez Andrés

2008-12-01

Full Text Available Abstract Background The elucidation of gene expression patterns leads to a better understanding of biological processes. Real-time quantitative RT-PCR has become the standard method for in-depth studies of gene expression. A biologically meaningful reporting of target mRNA quantities requires accurate and reliable normalization in order to identify real gene-specific variation. The purpose of normalization is to control several variables such as different amounts and quality of starting material, variable enzymatic efficiencies of retrotranscription from RNA to cDNA, or differences between tissues or cells in overall transcriptional activity. The validity of a housekeeping gene as endogenous control relies on the stability of its expression level across the sample panel being analysed. In the present report we describe the first systematic evaluation of potential internal controls during tomato development process to identify which are the most reliable for transcript quantification by real-time RT-PCR. Results In this study, we assess the expression stability of 7 traditional and 4 novel housekeeping genes in a set of 27 samples representing different tissues and organs of tomato plants at different developmental stages. First, we designed, tested and optimized amplification primers for real-time RT-PCR. Then, expression data from each candidate gene were evaluated with three complementary approaches based on different statistical procedures. Our analysis suggests that SGN-U314153 (CAC, SGN-U321250 (TIP41, SGN-U346908 ("Expressed" and SGN-U316474 (SAND genes provide superior transcript normalization in tomato development studies. We recommend different combinations of these exceptionally stable housekeeping genes for suited normalization of different developmental series, including the complete tomato development process. Conclusion This work constitutes the first effort for the selection of optimal endogenous controls for quantitative real

Identification of normalization factors for quantitative real-time RT-PCR analysis of gene expression in Pacific abalone Haliotis discus hannai

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Qiu, Reng; Sun, Boguang; Fang, Shasha; Sun, Li; Liu, Xiao

2013-03-01

Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is widely used in studies of gene expression. In most of these studies, housekeeping genes are used as internal references without validation. To identify appropriate reference genes for qRT-PCR in Pacific abalone Haliotis discus hannai, we examined the transcription stability of six housekeeping genes in abalone tissues in the presence and absence of bacterial infection. For this purpose, abalone were infected with the bacterial pathogen Vibrio anguillarum for 12 h and 48 h. The mRNA levels of the housekeeping genes in five tissues (digestive glands, foot muscle, gill, hemocyte, and mantle) were determined by qRT-PCR. The PCR data was subsequently analyzed with the geNorm and NormFinder algorithms. The results show that in the absence of bacterial infection, elongation factor-1-alpha and beta-actin were the most stably expressed genes in all tissues, and thus are suitable as cross-tissue type normalization factors. However, we did not identify any universal reference genes post infection because the most stable genes varied between tissue types. Furthermore, for most tissues, the optimal reference genes identified by both algorithms at 12 h and 48 h post-infection differed. These results indicate that bacterial infection induced significant changes in the expression of abalone housekeeping genes in a manner that is dependent on tissue type and duration of infection. As a result, different normalization factors must be used for different tissues at different infection points.

How to perform RT-qPCR accurately in plant species? A case study on flower colour gene expression in an azalea (Rhododendron simsii hybrids) mapping population.

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De Keyser, Ellen; Desmet, Laurence; Van Bockstaele, Erik; De Riek, Jan

2013-06-24

Flower colour variation is one of the most crucial selection criteria in the breeding of a flowering pot plant, as is also the case for azalea (Rhododendron simsii hybrids). Flavonoid biosynthesis was studied intensively in several species. In azalea, flower colour can be described by means of a 3-gene model. However, this model does not clarify pink-coloration. The last decade gene expression studies have been implemented widely for studying flower colour. However, the methods used were often only semi-quantitative or quantification was not done according to the MIQE-guidelines. We aimed to develop an accurate protocol for RT-qPCR and to validate the protocol to study flower colour in an azalea mapping population. An accurate RT-qPCR protocol had to be established. RNA quality was evaluated in a combined approach by means of different techniques e.g. SPUD-assay and Experion-analysis. We demonstrated the importance of testing noRT-samples for all genes under study to detect contaminating DNA. In spite of the limited sequence information available, we prepared a set of 11 reference genes which was validated in flower petals; a combination of three reference genes was most optimal. Finally we also used plasmids for the construction of standard curves. This allowed us to calculate gene-specific PCR efficiencies for every gene to assure an accurate quantification. The validity of the protocol was demonstrated by means of the study of six genes of the flavonoid biosynthesis pathway. No correlations were found between flower colour and the individual expression profiles. However, the combination of early pathway genes (CHS, F3H, F3'H and FLS) is clearly related to co-pigmentation with flavonols. The late pathway genes DFR and ANS are to a minor extent involved in differentiating between coloured and white flowers. Concerning pink coloration, we could demonstrate that the lower intensity in this type of flowers is correlated to the expression of F3'H. Currently in plant

Evaluation of candidate reference genes for gene expression normalization in Brassica juncea using real time quantitative RT-PCR.

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Ruby Chandna

Full Text Available The real time quantitative reverse transcription PCR (qRT-PCR is becoming increasingly important to gain insight into function of genes. Given the increased sensitivity, ease and reproducibility of qRT-PCR, the requirement of suitable reference genes for normalization has become important and stringent. It is now known that the expression of internal control genes in living organism vary considerably during developmental stages and under different experimental conditions. For economically important Brassica crops, only a couple of reference genes are reported till date. In this study, expression stability of 12 candidate reference genes including ACT2, ELFA, GAPDH, TUA, UBQ9 (traditional housekeeping genes, ACP, CAC, SNF, TIPS-41, TMD, TSB and ZNF (new candidate reference genes, in a diverse set of 49 tissue samples representing different developmental stages, stress and hormone treated conditions and cultivars of Brassica juncea has been validated. For the normalization of vegetative stages the ELFA, ACT2, CAC and TIPS-41 combination would be appropriate whereas TIPS-41 along with CAC would be suitable for normalization of reproductive stages. A combination of GAPDH, TUA, TIPS-41 and CAC were identified as the most suitable reference genes for total developmental stages. In various stress and hormone treated samples, UBQ9 and TIPS-41 had the most stable expression. Across five cultivars of B. juncea, the expression of CAC and TIPS-41 did not vary significantly and were identified as the most stably expressed reference genes. This study provides comprehensive information that the new reference genes selected herein performed better than the traditional housekeeping genes. The selection of most suitable reference genes depends on the experimental conditions, and is tissue and cultivar-specific. Further, to attain accuracy in the results more than one reference genes are necessary for normalization.

Validation of reference genes for RT-qPCR analysis of CYP4T expression in crucian carp

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Fei Mo

2014-09-01

Full Text Available Reference genes are commonly used for normalization of target gene expression during RT-qPCR analysis. However, no housekeeping genes or reference genes have been identified to be stable across different tissue types or under different experimental conditions. To identify the most suitable reference genes for RT-qPCR analysis of target gene expression in the hepatopancreas of crucian carp (Carassius auratus under various conditions (sex, age, water temperature, and drug treatments, seven reference genes, including beta actin (ACTB, beta-2 microglobulin (B2M, embryonic elongation factor-1 alpha (EEF1A, glyceraldehyde phosphate dehydrogenase (GAPDH, alpha tubulin (TUBA, ribosomal protein l8 (RPL8 and glucose-6-phosphate dehydrogenase (G6PDH, were evaluated in this study. The stability and ranking of gene expression were analyzed using three different statistical programs: GeNorm, Normfinder and Bestkeeper. The expression errors associated with selection of the genes were assessed by the relative quantity of CYP4T. The results indicated that all the seven genes exhibited variability under the experimental conditions of this research, and the combination of ACTB/TUBA/EEF1A or of ACTB/EEF1A was the best candidate that raised the accuracy of quantitative analysis of gene expression. The findings highlighted the importance of validation of housekeeping genes for research on gene expression under different conditions of experiment and species.

The workflow of single-cell expression profiling using quantitative real-time PCR

Czech Academy of Sciences Publication Activity Database

Stahlberg, A.; Kubista, Mikael

2014-01-01

Roč. 14, č. 3 (2014), s. 323-331 ISSN 1473-7159 R&D Projects: GA ČR GA13-02154S; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:86652036 Keywords : single-cell workflow * gene expression profiling * RT-qPCR Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.516, year: 2014

Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology.

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Smith, Cindy J; Osborn, A Mark

2009-01-01

Quantitative PCR (Q-PCR or real-time PCR) approaches are now widely applied in microbial ecology to quantify the abundance and expression of taxonomic and functional gene markers within the environment. Q-PCR-based analyses combine 'traditional' end-point detection PCR with fluorescent detection technologies to record the accumulation of amplicons in 'real time' during each cycle of the PCR amplification. By detection of amplicons during the early exponential phase of the PCR, this enables the quantification of gene (or transcript) numbers when these are proportional to the starting template concentration. When Q-PCR is coupled with a preceding reverse transcription reaction, it can be used to quantify gene expression (RT-Q-PCR). This review firstly addresses the theoretical and practical implementation of Q-PCR and RT-Q-PCR protocols in microbial ecology, highlighting key experimental considerations. Secondly, we review the applications of (RT)-Q-PCR analyses in environmental microbiology and evaluate the contribution and advances gained from such approaches. Finally, we conclude by offering future perspectives on the application of (RT)-Q-PCR in furthering understanding in microbial ecology, in particular, when coupled with other molecular approaches and more traditional investigations of environmental systems.

Selection of Housekeeping Genes for Transgene Expression Analysis in Eucommia ulmoides Oliver Using Real-Time RT-PCR

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Ren Chen

2010-01-01

Full Text Available In order to select appropriate housekeeping genes for accurate calibration of experimental variations in real-time (RT- PCR results in transgene expression analysis, particularly with respect to the influence of transgene on stability of endogenous housekeeping gene expression in transgenic plants, we outline a reliable strategy to identify the optimal housekeeping genes from a set of candidates by combining statistical analyses of their (RT- PCR amplification efficiency, gene expression stability, and transgene influences. We used the strategy to select two genes, ACTα and EF1α, from 10 candidate housekeeping genes, as the optimal housekeeping genes to evaluate transgenic Eucommia ulmoides Oliver root lines overexpressing IPPI or FPPS1 genes, which are involved in isoprenoid biosynthesis.

Selection and validation of reference genes for qRT-PCR expression analysis of candidate genes involved in olfactory communication in the butterfly Bicyclus anynana.

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Alok Arun

Full Text Available Real-time quantitative reverse transcription PCR (qRT-PCR is a technique widely used to quantify the transcriptional expression level of candidate genes. qRT-PCR requires the selection of one or several suitable reference genes, whose expression profiles remain stable across conditions, to normalize the qRT-PCR expression profiles of candidate genes. Although several butterfly species (Lepidoptera have become important models in molecular evolutionary ecology, so far no study aimed at identifying reference genes for accurate data normalization for any butterfly is available. The African bush brown butterfly Bicyclus anynana has drawn considerable attention owing to its suitability as a model for evolutionary ecology, and we here provide a maiden extensive study to identify suitable reference gene in this species. We monitored the expression profile of twelve reference genes: eEF-1α, FK506, UBQL40, RpS8, RpS18, HSP, GAPDH, VATPase, ACT3, TBP, eIF2 and G6PD. We tested the stability of their expression profiles in three different tissues (wings, brains, antennae, two developmental stages (pupal and adult and two sexes (male and female, all of which were subjected to two food treatments (food stress and control feeding ad libitum. The expression stability and ranking of twelve reference genes was assessed using two algorithm-based methods, NormFinder and geNorm. Both methods identified RpS8 as the best suitable reference gene for expression data normalization. We also showed that the use of two reference genes is sufficient to effectively normalize the qRT-PCR data under varying tissues and experimental conditions that we used in B. anynana. Finally, we tested the effect of choosing reference genes with different stability on the normalization of the transcript abundance of a candidate gene involved in olfactory communication in B. anynana, the Fatty Acyl Reductase 2, and we confirmed that using an unstable reference gene can drastically alter the

Selection and validation of reference genes for qRT-PCR expression analysis of candidate genes involved in olfactory communication in the butterfly Bicyclus anynana.

Science.gov (United States)

Arun, Alok; Baumlé, Véronique; Amelot, Gaël; Nieberding, Caroline M

2015-01-01

Real-time quantitative reverse transcription PCR (qRT-PCR) is a technique widely used to quantify the transcriptional expression level of candidate genes. qRT-PCR requires the selection of one or several suitable reference genes, whose expression profiles remain stable across conditions, to normalize the qRT-PCR expression profiles of candidate genes. Although several butterfly species (Lepidoptera) have become important models in molecular evolutionary ecology, so far no study aimed at identifying reference genes for accurate data normalization for any butterfly is available. The African bush brown butterfly Bicyclus anynana has drawn considerable attention owing to its suitability as a model for evolutionary ecology, and we here provide a maiden extensive study to identify suitable reference gene in this species. We monitored the expression profile of twelve reference genes: eEF-1α, FK506, UBQL40, RpS8, RpS18, HSP, GAPDH, VATPase, ACT3, TBP, eIF2 and G6PD. We tested the stability of their expression profiles in three different tissues (wings, brains, antennae), two developmental stages (pupal and adult) and two sexes (male and female), all of which were subjected to two food treatments (food stress and control feeding ad libitum). The expression stability and ranking of twelve reference genes was assessed using two algorithm-based methods, NormFinder and geNorm. Both methods identified RpS8 as the best suitable reference gene for expression data normalization. We also showed that the use of two reference genes is sufficient to effectively normalize the qRT-PCR data under varying tissues and experimental conditions that we used in B. anynana. Finally, we tested the effect of choosing reference genes with different stability on the normalization of the transcript abundance of a candidate gene involved in olfactory communication in B. anynana, the Fatty Acyl Reductase 2, and we confirmed that using an unstable reference gene can drastically alter the expression

Selection of reference genes for qRT-PCR analysis of gene expression in sea cucumber Apostichopus japonicus during aestivation

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Zhao, Ye; Chen, Muyan; Wang, Tianming; Sun, Lina; Xu, Dongxue; Yang, Hongsheng

2014-11-01

Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is a technique that is widely used for gene expression analysis, and its accuracy depends on the expression stability of the internal reference genes used as normalization factors. However, many applications of qRT-PCR used housekeeping genes as internal controls without validation. In this study, the expression stability of eight candidate reference genes in three tissues (intestine, respiratory tree, and muscle) of the sea cucumber Apostichopus japonicus was assessed during normal growth and aestivation using the geNorm, NormFinder, delta CT, and RefFinder algorithms. The results indicate that the reference genes exhibited significantly different expression patterns among the three tissues during aestivation. In general, the β-tubulin (TUBB) gene was relatively stable in the intestine and respiratory tree tissues. The optimal reference gene combination for intestine was 40S ribosomal protein S18 (RPS18), TUBB, and NADH dehydrogenase (NADH); for respiratory tree, it was β-actin (ACTB), TUBB, and succinate dehydrogenase cytochrome B small subunit (SDHC); and for muscle it was α-tubulin (TUBA) and NADH dehydrogenase [ubiquinone] 1 α subcomplex subunit 13 (NDUFA13). These combinations of internal control genes should be considered for use in further studies of gene expression in A. japonicus during aestivation.

Validation of reference genes for gene expression analysis in olive (Olea europaea) mesocarp tissue by quantitative real-time RT-PCR

Science.gov (United States)

2014-01-01

of expression stability across three different olive cultivars, Barnea, Frantoio and Picual, however the combination of the three most stable reference genes do vary amongst individual cultivars. This study will provide guidance to other researchers to select reference genes for normalization against target genes by qPCR across tissues obtained from the mesocarp region of the olive fruit in the cultivars, Barnea, Frantoio and Picual. PMID:24884716

Validation of reference genes for quantitative expression analysis by real-time rt-PCR in four lepidopteran insects.

Science.gov (United States)

Teng, Xiaolu; Zhang, Zan; He, Guiling; Yang, Liwen; Li, Fei

2012-01-01

Quantitative real-time polymerase chain reaction (qPCR) is an efficient and widely used technique to monitor gene expression. Housekeeping genes (HKGs) are often empirically selected as the reference genes for data normalization. However, the suitability of HKGs used as the reference genes has been seldom validated. Here, six HKGs were chosen (actin A3, actin A1, GAPDH, G3PDH, E2F, rp49) in four lepidopteran insects Bombyx mori L. (Lepidoptera: Bombycidae), Plutella xylostella L. (Plutellidae), Chilo suppressalis Walker (Crambidae), and Spodoptera exigua Hübner (Noctuidae) to study their expression stability. The algorithms of geNorm, NormFinder, stability index, and ΔCt analysis were used to evaluate these HKGs. Across different developmental stages, actin A1 was the most stable in P. xylostella and C. suppressalis, but it was the least stable in B. mori and S. exigua. Rp49 and GAPDH were the most stable in B. mori and S. exigua, respectively. In different tissues, GAPDH, E2F, and Rp49 were the most stable in B. mori, S. exigua, and C. suppressalis, respectively. The relative abundances of Siwi genes estimated by 2(-ΔΔCt) method were tested with different HKGs as the reference gene, proving the importance of internal controls in qPCR data analysis. The results not only presented a list of suitable reference genes in four lepidopteran insects, but also proved that the expression stabilities of HKGs were different among evolutionarily close species. There was no single universal reference gene that could be used in all situations. It is indispensable to validate the expression of HKGs before using them as the internal control in qPCR.

Expression analysis of fiber related genes in cotton (gossypium hirsutum l.) through real time pcr

International Nuclear Information System (INIS)

Iqbal, N.; Khatoon, A.; Asif, M.; Bashir, A.

2016-01-01

Cotton fibers are unicellular seed trichomes and the largest known plant cells. Fiber morphogenesis in cotton is a complex process involving a large number of genes expressed throughout fiber development process. The expression profiling of five gene families in various cotton tissues was carried out through real time PCR. Expression analysis revealed that transcripts of expansin, tubulin and E6 were elevated from 5 to 20 days post anthesis (DPA) fibers. Three Lipid transfer proteins (LTPs) including LTP1, LTP3, LTP7 exhibited highest expression in 10 - 20 DPA fibers. Transcripts of LTP3 were detected in fibers and non fiber tissues that of LTP7 were almost negligible in non fiber tissues. Sucrose phosphate synthase gene showed highest expression in 10 DPA fibers while sucrose synthse (susy) expressed at higher rate in 5-20 DPA fibers as well as roots. The results reveal that most of fiber related genes showed high expression in 5-20 DPA fibers. Comprehensive expression study may help to determine tissue and stage specificity of genes under study. The study may also help to explore complex process of fiber development and understand the role of these genes in fiber development process. Highly expressed genes in fibers may be transformed in cotton for improvement of fiber quality traits. Genes that were expressed specifically in fibers or other tissues could be used for isolation of upstream regulatory sequences. (author)

Screening Reliable Reference Genes for RT-qPCR Analysis of Gene Expression in Moringa oleifera.

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Deng, Li-Ting; Wu, Yu-Ling; Li, Jun-Cheng; OuYang, Kun-Xi; Ding, Mei-Mei; Zhang, Jun-Jie; Li, Shu-Qi; Lin, Meng-Fei; Chen, Han-Bin; Hu, Xin-Sheng; Chen, Xiao-Yang

2016-01-01

Moringa oleifera is a promising plant species for oil and forage, but its genetic improvement is limited. Our current breeding program in this species focuses on exploiting the functional genes associated with important agronomical traits. Here, we screened reliable reference genes for accurately quantifying the expression of target genes using the technique of real-time quantitative polymerase chain reaction (RT-qPCR) in M. oleifera. Eighteen candidate reference genes were selected from a transcriptome database, and their expression stabilities were examined in 90 samples collected from the pods in different developmental stages, various tissues, and the roots and leaves under different conditions (low or high temperature, sodium chloride (NaCl)- or polyethyleneglycol (PEG)- simulated water stress). Analyses with geNorm, NormFinder and BestKeeper algorithms revealed that the reliable reference genes differed across sample designs and that ribosomal protein L1 (RPL1) and acyl carrier protein 2 (ACP2) were the most suitable reference genes in all tested samples. The experiment results demonstrated the significance of using the properly validated reference genes and suggested the use of more than one reference gene to achieve reliable expression profiles. In addition, we applied three isotypes of the superoxide dismutase (SOD) gene that are associated with plant adaptation to abiotic stress to confirm the efficacy of the validated reference genes under NaCl and PEG water stresses. Our results provide a valuable reference for future studies on identifying important functional genes from their transcriptional expressions via RT-qPCR technique in M. oleifera.

Selection of Reliable Reference Genes for Gene Expression Studies in the Biofuel Plant Jatropha curcas Using Real-Time Quantitative PCR

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Lu Zhang

2013-12-01

Full Text Available Jatropha curcas is a promising renewable feedstock for biodiesel and bio-jet fuel production. To study gene expression in Jatropha in different tissues throughout development and under stress conditions, we examined a total of 11 typical candidate reference genes using real-time quantitative polymerase chain reaction (RT-qPCR analysis, which is widely used for validating transcript levels in gene expression studies. The expression stability of these candidate reference genes was assessed across a total of 20 samples, including various tissues at vegetative and reproductive stages and under desiccation and cold stress treatments. The results obtained using software qBasePLUS showed that the top-ranked reference genes differed across the sample subsets. The combination of actin, GAPDH, and EF1α would be appropriate as a reference panel for normalizing gene expression data across samples at different developmental stages; the combination of actin, GAPDH, and TUB5 should be used as a reference panel for normalizing gene expression data across samples under various abiotic stress treatments. With regard to different developmental stages, we recommend the use of actin and TUB8 for normalization at the vegetative stage and GAPDH and EF1α for normalization at the reproductive stage. For abiotic stress treatments, we recommend the use of TUB5 and TUB8 for normalization under desiccation stress and GAPDH and actin for normalization under cold stress. These results are valuable for future research on gene expression during development or under abiotic stress in Jatropha. To our knowledge, this is the first report on the stability of reference genes in Jatropha.

Evaluation and validation of candidate endogenous control genes for real-time quantitative PCR studies of breast cancer

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Miller Nicola

2007-11-01

Full Text Available Abstract Background Real-time quantitative PCR (RQ-PCR forms the basis of many breast cancer biomarker studies and novel prognostic assays, paving the way towards personalised cancer treatments. Normalisation of relative RQ-PCR data is required to control for non-biological variation introduced during sample preparation. Endogenous control (EC genes, used in this context, should ideally be expressed constitutively and uniformly across treatments in all test samples. Despite widespread recognition that the accuracy of the normalised data is largely dependent on the reliability of the EC, there are no reports of the systematic validation of genes commonly used for this purpose in the analysis of gene expression by RQ-PCR in primary breast cancer tissues. The aim of this study was to identify the most suitable endogenous control genes for RQ-PCR analysis of primary breast tissue from a panel of eleven candidates in current use. Oestrogen receptor alpha (ESR1 was used a target gene to compare the effect of choice of EC on the estimate of gene quantity. Results The expression and validity of candidate ECs (GAPDH, TFRC, ABL, PPIA, HPRT1, RPLP0, B2M, GUSB, MRPL19, PUM1 and PSMC4 was determined in 6 benign and 21 malignant primary breast cancer tissues. Gene expression data was analysed using two different statistical models. MRPL19 and PPIA were identified as the most stable and reliable EC genes, while GUSB, RPLP0 and ABL were least stable. There was a highly significant difference in variance between ECs. ESR1 expression was appreciably higher in malignant compared to benign tissues and there was a significant effect of EC on the magnitude of the error associated with the relative quantity of ESR1. Conclusion We have validated two endogenous control genes, MRPL19 and PPIA, for RQ-PCR analysis of gene expression in primary breast tissue. Of the genes in current use in this field, the above combination offers increased accuracy and resolution in the

SASqPCR: robust and rapid analysis of RT-qPCR data in SAS.

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Daijun Ling

Full Text Available Reverse transcription quantitative real-time PCR (RT-qPCR is a key method for measurement of relative gene expression. Analysis of RT-qPCR data requires many iterative computations for data normalization and analytical optimization. Currently no computer program for RT-qPCR data analysis is suitable for analytical optimization and user-controllable customization based on data quality, experimental design as well as specific research aims. Here I introduce an all-in-one computer program, SASqPCR, for robust and rapid analysis of RT-qPCR data in SAS. This program has multiple macros for assessment of PCR efficiencies, validation of reference genes, optimization of data normalizers, normalization of confounding variations across samples, and statistical comparison of target gene expression in parallel samples. Users can simply change the macro variables to test various analytical strategies, optimize results and customize the analytical processes. In addition, it is highly automatic and functionally extendable. Thus users are the actual decision-makers controlling RT-qPCR data analyses. SASqPCR and its tutorial are freely available at http://code.google.com/p/sasqpcr/downloads/list.

The normally expressed kappa immunoglobulin light chain gene repertoire and somatic mutations studied by single-sided specific polymerase chain reaction (PCR); frequent occurrence of features often assigned to autoimmunity

DEFF Research Database (Denmark)

Juul, L; Hougs, L; Andersen, V

1997-01-01

The expressed human kappa light chain gene repertoire utilized by healthy individuals was studied by two different single-sided specific PCR techniques to avoid bias for certain V genes. A total of 103 rearranged kappa sequences from peripheral blood mononuclear cells from healthy individuals were...

Assessment of brain reference genes for RT-qPCR studies in neurodegenerative diseases

OpenAIRE

Rydbirk, Rasmus; Folke, Jonas; Winge, Kristian; Aznar, Susana; Pakkenberg, Bente; Brudek, Tomasz

2016-01-01

Evaluation of gene expression levels by reverse transcription quantitative real-time PCR (RT-qPCR) has for many years been the favourite approach for discovering disease-associated alterations. Normalization of results to stably expressed reference genes (RGs) is pivotal to obtain reliable results. This is especially important in relation to neurodegenerative diseases where disease-related structural changes may affect the most commonly used RGs. We analysed 15 candidate RGs in 98 brain sampl...

Expression Stabilities of Ten Candidate Reference Genes for RT-qPCR in Zanthoxylum bungeanum Maxim.

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Fei, Xitong; Shi, Qianqian; Yang, Tuxi; Fei, Zhaoxue; Wei, Anzhi

2018-03-30

Real-time reverse transcription quantitative PCR has become a common method for studying gene expression, however, the optimal selection of stable reference genes is a prerequisite for obtaining accurate quantification of transcript abundance. Suitable reference genes for RT-qPCR have not yet been identified for Chinese prickly ash ( Zanthoxylum bungeanum Maxim.). Chinese prickly ash is the source of an important food seasoning in China. In recent years, Chinese prickly ash has also been developed as a medicinal plant. The expression stabilities of ten genes ( 18S , 28S , EF , UBA , UBQ , TIF , NTB , TUA , RPS , and TIF5A ) were evaluated in roots, stems, leaves, flowers and fruits at five developmental stages and also under stress from cold, drought, and salt. To do this we used three different statistical algorithms: geNorm, NormFinder and BestKeeper. Among the genes investigated, UBA and UBQ were found to be most stable for the different cultivars and different tissues examined, UBQ and TIF for fruit developmental stage. Meanwhile, EF and TUA were most stable under cold treatment, EF and UBQ under drought treatment and NTB and RPS under salt treatment. UBA and UBQ for all samples evaluated were most stably expressed, but 18S , TUA and RPS were found to be generally unreliable as reference genes. Our results provide a basis for the future selection of reference genes for biological research with Chinese prickly ash, under a variety of conditions.

Localisation of Abundant and Organ-Specific Genes Expressed in Rosa hybrida Leaves and Flower Buds by Direct In Situ RT-PCR

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Agata Jedrzejuk

2012-01-01

Full Text Available In situ PCR is a technique that allows specific nucleic acid sequences to be detected in individual cells and tissues. In situ PCR and IS-RT-PCR are elegant techniques that can increase both sensitivity and throughput, but they are, at best, only semiquantitative; therefore, it is desirable first to ascertain the expression pattern by conventional means to establish the suitable conditions for each probe. In plants, in situ RT-PCR is widely used in the expression localisation of specific genes, including MADS-box and other function-specific genes or housekeeping genes in floral buds and other organs. This method is especially useful in small organs or during early developmental stages when the separation of particular parts is impossible. In this paper, we compared three different labelling and immunodetection methods by using in situ RT-PCR in Rosa hybrida flower buds and leaves. As target genes, we used the abundant β-actin and RhFUL gene, which is expressed only in the leaves and petals/sepals of flower buds. We used digoxygenin-11-dUTP, biotin-11-dUTP, and fluorescein-12-dUTP-labelled nucleotides and antidig-AP/ streptavidin-fluorescein-labelled antibodies. All of the used methods gave strong, specific signal and all of them may be used in localization of gene expression on tissue level in rose organs.

Identification of suitable reference genes for gene expression normalization in qRT-PCR analysis in watermelon.

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Qiusheng Kong

Full Text Available Watermelon is one of the major Cucurbitaceae crops and the recent availability of genome sequence greatly facilitates the fundamental researches on it. Quantitative real-time reverse transcriptase PCR (qRT-PCR is the preferred method for gene expression analyses, and using validated reference genes for normalization is crucial to ensure the accuracy of this method. However, a systematic validation of reference genes has not been conducted on watermelon. In this study, transcripts of 15 candidate reference genes were quantified in watermelon using qRT-PCR, and the stability of these genes was compared using geNorm and NormFinder. geNorm identified ClTUA and ClACT, ClEF1α and ClACT, and ClCAC and ClTUA as the best pairs of reference genes in watermelon organs and tissues under normal growth conditions, abiotic stress, and biotic stress, respectively. NormFinder identified ClYLS8, ClUBCP, and ClCAC as the best single reference genes under the above experimental conditions, respectively. ClYLS8 and ClPP2A were identified as the best reference genes across all samples. Two to nine reference genes were required for more reliable normalization depending on the experimental conditions. The widely used watermelon reference gene 18SrRNA was less stable than the other reference genes under the experimental conditions. Catalase family genes were identified in watermelon genome, and used to validate the reliability of the identified reference genes. ClCAT1and ClCAT2 were induced and upregulated in the first 24 h, whereas ClCAT3 was downregulated in the leaves under low temperature stress. However, the expression levels of these genes were significantly overestimated and misinterpreted when 18SrRNA was used as a reference gene. These results provide a good starting point for reference gene selection in qRT-PCR analyses involving watermelon.

Quantification of low-expressed mRNA using 5' LNA-containing real-time PCR primers

International Nuclear Information System (INIS)

Malgoyre, A.; Banzet, S.; Mouret, C.; Bigard, A.X.; Peinnequin, A.

2007-01-01

Real-time RT-PCR is the most sensitive and accurate method for mRNA quantification. Using specific recombinant DNA as a template, real-time PCR allows accurate quantification within a 7-log range and increased sensitivity below 10 copies. However, when using RT-PCR to quantify mRNA in biological samples, a stochastic off-targeted amplification can occur. Classical adjustments of assay parameters have minimal effects on such amplification. This undesirable amplification appears mostly to be dependent on specific to non-specific target ratio rather than on the absolute quantity of the specific target. This drawback, which decreases assay reliability, mostly appears when quantifying low-expressed transcript in a whole organ. An original primer design using properties of LNA allows to block off-target amplification. 5'-LNA substitution strengthens 5'-hybridization. Consequently on-target hybridization is stabilized and the probability for the off-target to lead to amplification is decreased

Analysis of gene expression in small numbers of purified hemopoietic progenitor cells by RT-PCR.

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Ziegler, B L; Lamping, C P; Thoma, S J; Fliedner, T M

1995-05-01

Primitive hemopoietic stem cells represent the most probable targets for genetic alterations due to exposure to ionizing irradiation or chemical carcinogens. We have applied a two-step protocol for the purification of CD34+HLA-DR-/low hemopoietic progenitor cells from cord blood (CB). CD34+ cells were isolated by monoclonal antibody (mAb) against CD34 (My10) and immunomagnetic beads. Beads were cleaved off the CD34+ cells by enzymatic treatment with chymopapain. Due to chymopapain-resistance of epitopes recognized by the used mAbs purity control of CD34+ cells and separation into CD34+HLA-DR-/low and CD34+HLA-DR+ subsets could be performed by using flow cytometry. Two miniaturized procedures were applied to isolate poly(A)+ mRNA for the reverse transcription polymerase chain reaction (RT-PCR) from small numbers of CD34+HLA-DR-/low cells. In five experiments, the mean purity of immunomagnetically isolated CD34+ cells was 93.8% +/- 3.9. Flow cytometry sorting of CD34+ cells resulted in pure CD34+HLA-DR-/low populations (purity > 98.8%; range 98.8% to 99.9%; viability > 96%) with an average yield of 2600 +/- 800 cells/5 x 10(7) low density CB cells. By RT-PCR using both poly(A)+ mRNA isolation procedures, sequences corresponding to CD34 and beta 2-microglobulin were amplified from as few as 20 cells. Furthermore, a sequence-independent RT-PCR (SIP-RT-PCR) was applied to amplify the cDNA derived from five erythroblasts isolated from a burst-forming unit-erythroid (BFU-E). Upon hybridization, full-length c-fos message was detected in the SIP-RT-PCR amplified material. Our data demonstrate that gene expression can be detected at the transcriptional level in small numbers of hemopoietic progenitor cells. In addition, the SIP-RT-PCR may allow the amplification of unique mRNA species when subtractive hybridization procedures are performed. The presented data should be useful to analyze gene expression in rare subsets of radiation-exposed immature hemopoietic stem

Validating Internal Control Genes for the Accurate Normalization of qPCR Expression Analysis of the Novel Model Plant Setaria viridis.

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Julia Lambret-Frotté

Full Text Available Employing reference genes to normalize the data generated with quantitative PCR (qPCR can increase the accuracy and reliability of this method. Previous results have shown that no single housekeeping gene can be universally applied to all experiments. Thus, the identification of a suitable reference gene represents a critical step of any qPCR analysis. Setaria viridis has recently been proposed as a model system for the study of Panicoid grasses, a crop family of major agronomic importance. Therefore, this paper aims to identify suitable S. viridis reference genes that can enhance the analysis of gene expression in this novel model plant. The first aim of this study was the identification of a suitable RNA extraction method that could retrieve a high quality and yield of RNA. After this, two distinct algorithms were used to assess the gene expression of fifteen different candidate genes in eighteen different samples, which were divided into two major datasets, the developmental and the leaf gradient. The best-ranked pair of reference genes from the developmental dataset included genes that encoded a phosphoglucomutase and a folylpolyglutamate synthase; genes that encoded a cullin and the same phosphoglucomutase as above were the most stable genes in the leaf gradient dataset. Additionally, the expression pattern of two target genes, a SvAP3/PI MADS-box transcription factor and the carbon-fixation enzyme PEPC, were assessed to illustrate the reliability of the chosen reference genes. This study has shown that novel reference genes may perform better than traditional housekeeping genes, a phenomenon which has been previously reported. These results illustrate the importance of carefully validating reference gene candidates for each experimental set before employing them as universal standards. Additionally, the robustness of the expression of the target genes may increase the utility of S. viridis as a model for Panicoid grasses.

Multiplex enrichment quantitative PCR (ME-qPCR): a high-throughput, highly sensitive detection method for GMO identification.

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Fu, Wei; Zhu, Pengyu; Wei, Shuang; Zhixin, Du; Wang, Chenguang; Wu, Xiyang; Li, Feiwu; Zhu, Shuifang

2017-04-01

Among all of the high-throughput detection methods, PCR-based methodologies are regarded as the most cost-efficient and feasible methodologies compared with the next-generation sequencing or ChIP-based methods. However, the PCR-based methods can only achieve multiplex detection up to 15-plex due to limitations imposed by the multiplex primer interactions. The detection throughput cannot meet the demands of high-throughput detection, such as SNP or gene expression analysis. Therefore, in our study, we have developed a new high-throughput PCR-based detection method, multiplex enrichment quantitative PCR (ME-qPCR), which is a combination of qPCR and nested PCR. The GMO content detection results in our study showed that ME-qPCR could achieve high-throughput detection up to 26-plex. Compared to the original qPCR, the Ct values of ME-qPCR were lower for the same group, which showed that ME-qPCR sensitivity is higher than the original qPCR. The absolute limit of detection for ME-qPCR could achieve levels as low as a single copy of the plant genome. Moreover, the specificity results showed that no cross-amplification occurred for irrelevant GMO events. After evaluation of all of the parameters, a practical evaluation was performed with different foods. The more stable amplification results, compared to qPCR, showed that ME-qPCR was suitable for GMO detection in foods. In conclusion, ME-qPCR achieved sensitive, high-throughput GMO detection in complex substrates, such as crops or food samples. In the future, ME-qPCR-based GMO content identification may positively impact SNP analysis or multiplex gene expression of food or agricultural samples. Graphical abstract For the first-step amplification, four primers (A, B, C, and D) have been added into the reaction volume. In this manner, four kinds of amplicons have been generated. All of these four amplicons could be regarded as the target of second-step PCR. For the second-step amplification, three parallels have been taken for

Selection and evaluation of potential reference genes for gene expression analysis in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae using reverse-transcription quantitative PCR.

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Miao Yuan

Full Text Available The brown planthopper (BPH, Nilaparvata lugens (Hemiptera, Delphacidae, is one of the most important rice pests. Abundant genetic studies on BPH have been conducted using reverse-transcription quantitative real-time PCR (qRT-PCR. Using qRT-PCR, the expression levels of target genes are calculated on the basis of endogenous controls. These genes need to be appropriately selected by experimentally assessing whether they are stably expressed under different conditions. However, such studies on potential reference genes in N. lugens are lacking. In this paper, we presented a systematic exploration of eight candidate reference genes in N. lugens, namely, actin 1 (ACT, muscle actin (MACT, ribosomal protein S11 (RPS11, ribosomal protein S15e (RPS15, alpha 2-tubulin (TUB, elongation factor 1 delta (EF, 18S ribosomal RNA (18S, and arginine kinase (AK and used four alternative methods (BestKeeper, geNorm, NormFinder, and the delta Ct method to evaluate the suitability of these genes as endogenous controls. We examined their expression levels among different experimental factors (developmental stage, body part, geographic population, temperature variation, pesticide exposure, diet change, and starvation following the MIQE (Minimum Information for publication of Quantitative real time PCR Experiments guidelines. Based on the results of RefFinder, which integrates four currently available major software programs to compare and rank the tested candidate reference genes, RPS15, RPS11, and TUB were found to be the most suitable reference genes in different developmental stages, body parts, and geographic populations, respectively. RPS15 was the most suitable gene under different temperature and diet conditions, while RPS11 was the most suitable gene under different pesticide exposure and starvation conditions. This work sheds light on establishing a standardized qRT-PCR procedure in N. lugens, and serves as a starting point for screening for reference genes for

Multiway real-time PCR gene expression profiling in yeast Saccharomyces cerevisiae reveals altered transcriptional response of ADH-genes to glucose stimuli.

Science.gov (United States)

Ståhlberg, Anders; Elbing, Karin; Andrade-Garda, José Manuel; Sjögreen, Björn; Forootan, Amin; Kubista, Mikael

2008-04-16

The large sensitivity, high reproducibility and essentially unlimited dynamic range of real-time PCR to measure gene expression in complex samples provides the opportunity for powerful multivariate and multiway studies of biological phenomena. In multiway studies samples are characterized by their expression profiles to monitor changes over time, effect of treatment, drug dosage etc. Here we perform a multiway study of the temporal response of four yeast Saccharomyces cerevisiae strains with different glucose uptake rates upon altered metabolic conditions. We measured the expression of 18 genes as function of time after addition of glucose to four strains of yeast grown in ethanol. The data are analyzed by matrix-augmented PCA, which is a generalization of PCA for 3-way data, and the results are confirmed by hierarchical clustering and clustering by Kohonen self-organizing map. Our approach identifies gene groups that respond similarly to the change of nutrient, and genes that behave differently in mutant strains. Of particular interest is our finding that ADH4 and ADH6 show a behavior typical of glucose-induced genes, while ADH3 and ADH5 are repressed after glucose addition. Multiway real-time PCR gene expression profiling is a powerful technique which can be utilized to characterize functions of new genes by, for example, comparing their temporal response after perturbation in different genetic variants of the studied subject. The technique also identifies genes that show perturbed expression in specific strains.

Multiway real-time PCR gene expression profiling in yeast Saccharomyces cerevisiae reveals altered transcriptional response of ADH-genes to glucose stimuli

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Andrade-Garda José

2008-04-01

Full Text Available Abstract Background The large sensitivity, high reproducibility and essentially unlimited dynamic range of real-time PCR to measure gene expression in complex samples provides the opportunity for powerful multivariate and multiway studies of biological phenomena. In multiway studies samples are characterized by their expression profiles to monitor changes over time, effect of treatment, drug dosage etc. Here we perform a multiway study of the temporal response of four yeast Saccharomyces cerevisiae strains with different glucose uptake rates upon altered metabolic conditions. Results We measured the expression of 18 genes as function of time after addition of glucose to four strains of yeast grown in ethanol. The data are analyzed by matrix-augmented PCA, which is a generalization of PCA for 3-way data, and the results are confirmed by hierarchical clustering and clustering by Kohonen self-organizing map. Our approach identifies gene groups that respond similarly to the change of nutrient, and genes that behave differently in mutant strains. Of particular interest is our finding that ADH4 and ADH6 show a behavior typical of glucose-induced genes, while ADH3 and ADH5 are repressed after glucose addition. Conclusion Multiway real-time PCR gene expression profiling is a powerful technique which can be utilized to characterize functions of new genes by, for example, comparing their temporal response after perturbation in different genetic variants of the studied subject. The technique also identifies genes that show perturbed expression in specific strains.

Development of FQ-PCR method to determine the level of ADD1 expression in fatty and lean pigs.

Science.gov (United States)

Cui, J X; Chen, W; Zeng, Y Q

2015-10-30

To determine how adipocyte determination and differentiation factor 1 (ADD1), a gene involved in the determination of pork quality, is regulated in Laiwu and Large pigs, we used TaqMan fluorescence quantitative real-time polymerase chain reaction (FQ-PCR) to detect differential expression in the longissimus muscle of Laiwu (fatty) and Large White (lean) pigs. In this study, the ADD1 and GAPDH cDNA sequences were cloned using a T-A cloning assay, and the clone sequences were consistent with those deposited in GenBank. Thus, the target fragment was successfully recombined into the vector, and its integrity was maintained. The standard curve and regression equation were established through the optimized FQ-PCR protocol. The standard curve of porcine ADD1 and GAPDH cDNA was determined, and its linear range extension could reach seven orders of magnitudes. The results showed that this method was used to quantify ADD1 expression in the longissimus muscle of two breeds of pig, and was found to be accurate, sensitive, and convenient. These results provide information regarding porcine ADD1 mRNA expression and the mechanism of adipocyte differentiation, and this study could help in the effort to meet the demands of consumers interested in the maintenance of health and prevention of obesity. Furthermore, it could lead to new approaches in the prevention and clinical treatment of this disease.

Housekeeping gene expression during fetal brain development in the rat-validation by semi-quantitative RT-PCR.

Science.gov (United States)

Al-Bader, Maie Dawoud; Al-Sarraf, Hameed Ali

2005-04-21

Mammalian gene expression is usually carried out at the level of mRNA where the amount of mRNA of interest is measured under different conditions such as growth and development. It is therefore important to use a "housekeeping gene", that does not change in relative abundance during the experimental conditions, as a standard or internal control. However, recent data suggest that expression of some housekeeping genes may vary with the extent of cell proliferation, differentiation and under various experimental conditions. In this study, the expression of various housekeeping genes (18S rRNA [18S], glyceraldehydes-3-phosphate dehydrogenase [G3PDH], beta-glucuronidase [BGLU], histone H4 [HH4], ribosomal protein L19 [RPL19] and cyclophilin [CY]) was investigated during fetal rat brain development using semi-quantitative RT-PCR at 16, 19 and 21 days gestation. It was found that all genes studied, with exception to G3PDH, did not show any change in their expression levels during development. G3PDH, on the other hand, showed increased expression with development. These results suggest that the choice of a housekeeping gene is critical to the interpretation of experimental results and should be modified according to the nature of the study.

Cytoglobin expression is upregulated in all tissues upon hypoxia: an in vitro and in vivo study by quantitative real-time PCR

International Nuclear Information System (INIS)

Fordel, E.; Geuens, E.; Dewilde, S.; Rottiers, P.; Carmeliet, P.; Grooten, J.; Moens, L.

2004-01-01

The vertebrate globin family has been extended with two members: neuroglobin and cytoglobin. We here investigate the changes of expression levels upon hypoxia of cytoglobin in parallel with neuroglobin, in vivo and in vitro, by using real-time quantitative PCR. Our data prove that cytoglobin is upregulated upon hypoxia in all tissues. The mechanism of induction of cytoglobin is regulated by the hypoxia-inducible factor 1, a posttranscriptionally regulated transcription factor controlling several hypoxia-inducible genes. The latter is argumented by: (1) cytoglobin is significantly upregulated upon hypoxia and this is dependent on the tissue and severity of hypoxia; (2) the regulation of cytoglobin expression in HIF-1 (+/-) knockout mice is affected; (3) the variations of the expression regulation are in the same manner as seen in the expression of our control gene VEGF, that is proven to be regulated by the HIF-1-pathway; and (4) cytoglobin promoter region contains HRE sites

Pre-amplification in the context of high-throughput qPCR gene expression experiment

Czech Academy of Sciences Publication Activity Database

Korenková, Vlasta; Scott, J.; Novosadová, Vendula; Jindřichová, Marie; Langerová, Lucie; Švec, David; Šídová, Monika; Sjoback, R.

2015-01-01

Roč. 16, č. 5 (2015) ISSN 1471-2199 R&D Projects: GA ČR(CZ) GAP304/12/1585; GA ČR(CZ) GA15-08239S; GA ČR GA13-02154S; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:86652036 Keywords : High-throughput qPCR * Gene expression * Exponential pre-amplification Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.500, year: 2015

Comparison of lung cancer cell lines representing four histopathological subtypes with gene expression profiling using quantitative real-time PCR

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Kawaguchi Makoto

2010-01-01

Full Text Available Abstract Background Lung cancers are the most common type of human malignancy and are intractable. Lung cancers are generally classified into four histopathological subtypes: adenocarcinoma (AD, squamous cell carcinoma (SQ, large cell carcinoma (LC, and small cell carcinoma (SC. Molecular biological characterization of these subtypes has been performed mainly using DNA microarrays. In this study, we compared the gene expression profiles of these four subtypes using twelve human lung cancer cell lines and the more reliable quantitative real-time PCR (qPCR. Results We selected 100 genes from public DNA microarray data and examined them by DNA microarray analysis in eight test cell lines (A549, ABC-1, EBC-1, LK-2, LU65, LU99, STC 1, RERF-LC-MA and a normal control lung cell line (MRC-9. From this, we extracted 19 candidate genes. We quantified the expression of the 19 genes and a housekeeping gene, GAPDH, with qPCR, using the same eight cell lines plus four additional validation lung cancer cell lines (RERF-LC-MS, LC-1/sq, 86-2, and MS-1-L. Finally, we characterized the four subtypes of lung cancer cell lines using principal component analysis (PCA of gene expression profiling for 12 of the 19 genes (AMY2A, CDH1, FOXG1, IGSF3, ISL1, MALL, PLAU, RAB25, S100P, SLCO4A1, STMN1, and TGM2. The combined PCA and gene pathway analyses suggested that these genes were related to cell adhesion, growth, and invasion. S100P in AD cells and CDH1 in AD and SQ cells were identified as candidate markers of these lung cancer subtypes based on their upregulation and the results of PCA analysis. Immunohistochemistry for S100P and RAB25 was closely correlated to gene expression. Conclusions These results show that the four subtypes, represented by 12 lung cancer cell lines, were well characterized using qPCR and PCA for the 12 genes examined. Certain genes, in particular S100P and CDH1, may be especially important for distinguishing the different subtypes. Our results

ReadqPCR and NormqPCR: R packages for the reading, quality checking and normalisation of RT-qPCR quantification cycle (Cq data

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Perkins James R

2012-07-01

Full Text Available Abstract Background Measuring gene transcription using real-time reverse transcription polymerase chain reaction (RT-qPCR technology is a mainstay of molecular biology. Technologies now exist to measure the abundance of many transcripts in parallel. The selection of the optimal reference gene for the normalisation of this data is a recurring problem, and several algorithms have been developed in order to solve it. So far nothing in R exists to unite these methods, together with other functions to read in and normalise the data using the chosen reference gene(s. Results We have developed two R/Bioconductor packages, ReadqPCR and NormqPCR, intended for a user with some experience with high-throughput data analysis using R, who wishes to use R to analyse RT-qPCR data. We illustrate their potential use in a workflow analysing a generic RT-qPCR experiment, and apply this to a real dataset. Packages are available from http://www.bioconductor.org/packages/release/bioc/html/ReadqPCR.htmland http://www.bioconductor.org/packages/release/bioc/html/NormqPCR.html Conclusions These packages increase the repetoire of RT-qPCR analysis tools available to the R user and allow them to (amongst other things read their data into R, hold it in an ExpressionSet compatible R object, choose appropriate reference genes, normalise the data and look for differential expression between samples.

Application of anti-listerial bacteriocins: monitoring enterocin expression by multiplex relative reverse transcription-PCR.

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Williams, D Ross; Chanos, Panagiotis

2012-12-01

Listeriosis is a deadly food-borne disease, and its incidence may be limited through the biotechnological exploitation of a number of anti-listerial biocontrol agents. The most widely used of these agents are bacteriocins and the Class II enterocins are characterized by their activity against Listeria. Enterocins are primarily produced by enterococci, particularly Enterococcus faecium and many strains have been described, often encoding multiple bacteriocins. The use of these strains in food will require that they are free of virulence functions and that they exhibit a high level expression of anti-listerial enterocins in fermentation conditions. Multiplex relative RT (reverse transcription)-PCR is a technique that is useful in the discovery of advantageous expression characteristics among enterocin-producing strains. It allows the levels of individual enterocin gene expression to be monitored and determination of how expression is altered under different growth conditions.

Expression analysis of fusarium wilt resistance gene in melon by real-time quantitative pcr

International Nuclear Information System (INIS)

Wang, X.; Xu, B.; Zhao, L.; Gao, P.; Luan, F.

2014-01-01

Melon Actin gene was used as a reference gene, to explore the gene expression profiles of the Fom-2 gene in roots, stems, and leaves of melon MR-1 under induction by Fusarium oxysporum f. sp. melonis. Monitoring using real-time quantitative PCR showed similar accumulation patterns of Fom-2 in roots, stems, and leaves over the observation period of 1 to 11 days; the expression level in stems was the highest. The expression of the Fom-2 gene was strengthened by the prolongation of induction time. In stems, the expression of Fom-2 was 5.737 times higher than in the control at three days; in roots, expression of Fom-2 was 5.617 times higher than in the control at five days. Similarly, the expression of Fom-2 in leaves obviously increased. It was 4.441 times higher than in the control at 5 days. The expression of Fom-2 was non-tissue specific, up-regulated under induction by Fusarium, and related to early resistance to Fusarium wilt. (author)

RT-PCR detection of Candida albicans ALS gene expression in the reconstituted human epithelium (RHE) model of oral candidiasis and in model biofilms.

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Green, Clayton B; Cheng, Georgina; Chandra, Jyotsna; Mukherjee, Pranab; Ghannoum, Mahmoud A; Hoyer, Lois L

2004-02-01

An RT-PCR assay was developed to analyse expression patterns of genes in the Candida albicans ALS (agglutinin-like sequence) family. Inoculation of a reconstituted human buccal epithelium (RHE) model of mucocutaneous candidiasis with strain SC5314 showed destruction of the epithelial layer by C. albicans and also formation of an upper fungal layer that had characteristics similar to a biofilm. RT-PCR analysis of total RNA samples extracted from C. albicans-inoculated buccal RHE showed that ALS1, ALS2, ALS3, ALS4, ALS5 and ALS9 were consistently detected over time as destruction of the RHE progressed. Detection of transcripts from ALS7, and particularly from ALS6, was more sporadic, but not associated with a strictly temporal pattern. The expression pattern of ALS genes in C. albicans cultures used to inoculate the RHE was similar to that observed in the RHE model, suggesting that contact of C. albicans with buccal RHE does little to alter ALS gene expression. RT-PCR analysis of RNA samples extracted from model denture and catheter biofilms showed similar gene expression patterns to the buccal RHE specimens. Results from the RT-PCR analysis of biofilm RNA specimens were consistent between various C. albicans strains during biofilm development and were comparable to gene expression patterns in planktonic cells. The RT-PCR assay described here will be useful for analysis of human clinical specimens and samples from other disease models. The method will provide further insight into the role of ALS genes and their encoded proteins in the diverse interactions between C. albicans and its host.

Evaluation of gamma-sterilization (60Co) by RT-PCR by DHFR expression detection

International Nuclear Information System (INIS)

Converso, Ana Paula G.; Andrade Junior, Heitor F. de; Vieira, Daniel P.

2007-01-01

The improvement of techniques to detect pathogen agents in blood had reduced significantly the contamination mechanisms by hemocomponents in blood transfusion procedures. Ionizing radiation is a method that has presented several applications on medicine and in currently days has been showing special attention on blood banks which has been applied to avoid TA-GVHD development. DHFR is an enzyme constitutive in Plasmodium protozoa and has an important role in folate metabolism on these parasites. Detecting the expression of RNAm coder for this enzyme is possible to evaluate the viability of this parasite in blood samples. Plasmodium chabaudi AJ is a parasite that induces lethal malaria in rodents similar to human malaria In this work, the objective was to detect the presence of plasmodium protozoa in irradiated blood samples, infected experimentally, through the application of a RT-PCR using primers for the coder sequence of DHFR's mRNA. We studied doses of ionizing radiation between 0 and 75 Gy. The irradiation procedures were accomplished in Center of Radiation Technology of IPEN-CNEN in a 60 Co panoramic source. Our results had demonstrated that RT-PCR is a sensible method to evaluate the viability of plasmodium in blood samples because the technique could detect low parasite burden in all tested samples. (author)

Real-time PCR (qPCR) primer design using free online software.

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Thornton, Brenda; Basu, Chhandak

2011-01-01

Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most commonly used fluorescent chemistries are SYBR® Green dyes and TaqMan®, Molecular Beacon or Scorpion probes. SYBR® Green is very simple to use and cost efficient. As SYBR® Green dye binds to any double-stranded DNA product, its success depends greatly on proper primer design. Many types of online primer design software are available, which can be used free of charge to design desirable SYBR® Green-based qPCR primers. This laboratory exercise is intended for those who have a fundamental background in PCR. It addresses the basic fluorescent chemistries of real-time PCR, the basic rules and pitfalls of primer design, and provides a step-by-step protocol for designing SYBR® Green-based primers with free, online software. Copyright © 2010 Wiley Periodicals, Inc.

Selection of reference genes for expression analysis using quantitative real-time PCR in the pea aphid, Acyrthosiphon pisum (Harris (Hemiptera, Aphidiae.

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Chunxiao Yang

Full Text Available To facilitate gene expression study and obtain accurate qRT-PCR analysis, normalization relative to stable expressed housekeeping genes is required. In this study, expression profiles of 11 candidate reference genes, including actin (Actin, elongation factor 1 α (EF1A, TATA-box-binding protein (TATA, ribosomal protein L12 (RPL12, β-tubulin (Tubulin, NADH dehydrogenase (NADH, vacuolar-type H+-ATPase (v-ATPase, succinate dehydrogenase B (SDHB, 28S ribosomal RNA (28S, 16S ribosomal RNA (16S, and 18S ribosomal RNA (18S from the pea aphid Acyrthosiphon pisum, under different developmental stages and temperature conditions, were investigated. A total of four analytical tools, geNorm, Normfinder, BestKeeper, and the ΔCt method, were used to evaluate the suitability of these genes as endogenous controls. According to RefFinder, a web-based software tool which integrates all four above-mentioned algorithms to compare and rank the reference genes, SDHB, 16S, and NADH were the three most stable house-keeping genes under different developmental stages and temperatures. This work is intended to establish a standardized qRT-PCR protocol in pea aphid and serves as a starting point for the genomics and functional genomics research in this emerging insect model.

Reliable gene expression analysis by reverse transcription-quantitative PCR: reporting and minimizing the uncertainty in data accuracy.

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Remans, Tony; Keunen, Els; Bex, Geert Jan; Smeets, Karen; Vangronsveld, Jaco; Cuypers, Ann

2014-10-01

Reverse transcription-quantitative PCR (RT-qPCR) has been widely adopted to measure differences in mRNA levels; however, biological and technical variation strongly affects the accuracy of the reported differences. RT-qPCR specialists have warned that, unless researchers minimize this variability, they may report inaccurate differences and draw incorrect biological conclusions. The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines describe procedures for conducting and reporting RT-qPCR experiments. The MIQE guidelines enable others to judge the reliability of reported results; however, a recent literature survey found low adherence to these guidelines. Additionally, even experiments that use appropriate procedures remain subject to individual variation that statistical methods cannot correct. For example, since ideal reference genes do not exist, the widely used method of normalizing RT-qPCR data to reference genes generates background noise that affects the accuracy of measured changes in mRNA levels. However, current RT-qPCR data reporting styles ignore this source of variation. In this commentary, we direct researchers to appropriate procedures, outline a method to present the remaining uncertainty in data accuracy, and propose an intuitive way to select reference genes to minimize uncertainty. Reporting the uncertainty in data accuracy also serves for quality assessment, enabling researchers and peer reviewers to confidently evaluate the reliability of gene expression data. © 2014 American Society of Plant Biologists. All rights reserved.

Development of a new comprehensive and reliable endometrial receptivity map (ER Map/ER Grade) based on RT-qPCR gene expression analysis.

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Enciso, M; Carrascosa, J P; Sarasa, J; Martínez-Ortiz, P A; Munné, S; Horcajadas, J A; Aizpurua, J

2018-02-01

Is it possible to determine the receptivity status of an endometrium by combined quantitative reverse transcription PCR (RT-qPCR) expression analysis of genes involved in endometrial proliferation and immunity? The new ER Map®/ER Grade® test can predict endometrial receptivity status by RT-qPCR using a new panel of genes involved in endometrial proliferation and the maternal immune response associated to embryonic implantation. The human endometrium reaches a receptive status adequate for embryonic implantation around Days 19-21 of the menstrual cycle. During this period, known as the window of implantation (WOI), the endometrium shows a specific gene expression profile suitable for endometrial function evaluation. The number of molecular diagnostic tools currently available to characterize this process is very limited. In this study, a new system for human endometrial receptivity evaluation was optimized and presented for the first time. ER Map®/ER Grade® validation was achieved on 312 endometrial samples including fertile women and patients undergoing fertility treatment between July 2014 and March 2016. Expression analyses of 184 genes involved in endometrial receptivity and immune response were performed. Samples were additionally tested with an independent endometrial receptivity test. A total of 96 fertile women and 120 assisted reproduction treatment (ART) patients participated in the study. Endometrial biopsy samples were obtained at LH + 2 and LH + 7 days in fertile subjects in a natural cycle and at the window of implantation (WOI) in patients in a hormone-replacement therapy (HRT) cycle. Total RNA was purified, quality-checked and reverse-transcribed. Gene expression was quantified by high-throughput RT-qPCR and statistically analyzed. Informative genes were selected and used to classify samples into four different groups of endometrial receptivity status. Significantly different gene expression levels were found in 85 out of 184 selected genes when

Gene expression profiling of acute myeloid leukemia samples from adult patients with AML-M1 and -M2 through boutique microarrays, real-time PCR and droplet digital PCR.

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Handschuh, Luiza; Kaźmierczak, Maciej; Milewski, Marek C; Góralski, Michał; Łuczak, Magdalena; Wojtaszewska, Marzena; Uszczyńska-Ratajczak, Barbara; Lewandowski, Krzysztof; Komarnicki, Mieczysław; Figlerowicz, Marek

2018-03-01

Acute myeloid leukemia (AML) is the most common and severe form of acute leukemia diagnosed in adults. Owing to its heterogeneity, AML is divided into classes associated with different treatment outcomes and specific gene expression profiles. Based on previous studies on AML, in this study, we designed and generated an AML-array containing 900 oligonucleotide probes complementary to human genes implicated in hematopoietic cell differentiation and maturation, proliferation, apoptosis and leukemic transformation. The AML-array was used to hybridize 118 samples from 33 patients with AML of the M1 and M2 subtypes of the French-American‑British (FAB) classification and 15 healthy volunteers (HV). Rigorous analysis of the microarray data revealed that 83 genes were differentially expressed between the patients with AML and the HV, including genes not yet discussed in the context of AML pathogenesis. The most overexpressed genes in AML were STMN1, KITLG, CDK6, MCM5, KRAS, CEBPA, MYC, ANGPT1, SRGN, RPLP0, ENO1 and SET, whereas the most underexpressed genes were IFITM1, LTB, FCN1, BIRC3, LYZ, ADD3, S100A9, FCER1G, PTRPE, CD74 and TMSB4X. The overexpression of the CPA3 gene was specific for AML with mutated NPM1 and FLT3. Although the microarray-based method was insufficient to differentiate between any other AML subgroups, quantitative PCR approaches enabled us to identify 3 genes (ANXA3, S100A9 and WT1) whose expression can be used to discriminate between the 2 studied AML FAB subtypes. The expression levels of the ANXA3 and S100A9 genes were increased, whereas those of WT1 were decreased in the AML-M2 compared to the AML-M1 group. We also examined the association between the STMN1, CAT and ABL1 genes, and the FLT3 and NPM1 mutation status. FLT3+/NPM1- AML was associated with the highest expression of STMN1, and ABL1 was upregulated in FLT3+ AML and CAT in FLT3- AML, irrespectively of the NPM1 mutation status. Moreover, our results indicated that CAT and WT1

Selection of Reference Genes for qRT-PCR Analysis of Gene Expression in Stipa grandis during Environmental Stresses.

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Dongli Wan

Full Text Available Stipa grandis P. Smirn. is a dominant plant species in the typical steppe of the Xilingole Plateau of Inner Mongolia. Selection of suitable reference genes for the quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR is important for gene expression analysis and research into the molecular mechanisms underlying the stress responses of S. grandis. In the present study, 15 candidate reference genes (EF1 beta, ACT, GAPDH, SamDC, CUL4, CAP, SNF2, SKIP1, SKIP5, SKIP11, UBC2, UBC15, UBC17, UCH, and HERC2 were evaluated for their stability as potential reference genes for qRT-PCR under different stresses. Four algorithms were used: GeNorm, NormFinder, BestKeeper, and RefFinder. The results showed that the most stable reference genes were different under different stress conditions: EF1beta and UBC15 during drought and salt stresses; ACT and GAPDH under heat stress; SKIP5 and UBC17 under cold stress; UBC15 and HERC2 under high pH stress; UBC2 and UBC15 under wounding stress; EF1beta and UBC17 under jasmonic acid treatment; UBC15 and CUL4 under abscisic acid treatment; and HERC2 and UBC17 under salicylic acid treatment. EF1beta and HERC2 were the most suitable genes for the global analysis of all samples. Furthermore, six target genes, SgPOD, SgPAL, SgLEA, SgLOX, SgHSP90 and SgPR1, were selected to validate the most and least stable reference genes under different treatments. Our results provide guidelines for reference gene selection for more accurate qRT-PCR quantification and will promote studies of gene expression in S. grandis subjected to environmental stress.

Selection of reference genes for quantitative real-time RT-PCR studies in tomato fruit of the genotype MT-Rg1

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Karla L. González-Aguilera

2016-09-01

Full Text Available Quantitative real-time RT-PCR (qRT-PCR has become one of the most widely used methods for accurate quantification of gene expression. Since there are no universal reference genes for normalization, the optimal strategy to normalize raw qRT-PCR data is to perform an initial comparison of a set of independent reference genes to assess the most stable ones in each biological model. Normalization of a qRT-PCR experiment helps to ensure that the results are both statistically significant and biologically meaningful. Tomato is the model of choice to study fleshy fruit development. The miniature tomato (Solanum lycopersicum L. cultivar Micro-Tom (MT is considered a model system for tomato genetics and functional genomics. A new genotype, containing the Rg1 allele, improves tomato in vitro regeneration. In this work, we evaluated the expression stability of four tomato reference genes, namely CAC, SAND, Expressed and ACTIN2. We showed that the genes CAC and Exp are the best reference genes of the four we tested during fruit development in the MT-Rg1 genotype. Furthermore, we validated the reference genes by showing that the expression profiles of the transcription factors FRUITFULL1 (FUL1 and APETALA2c (AP2c during fruit development are comparable to previous reports using other tomato cultivars.

Evaluation of gamma-sterilization ({sup 60}Co) by RT-PCR by DHFR expression detection

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Converso, Ana Paula G.; Andrade Junior, Heitor F. de [Instituto de Medicina Tropical de Sao Paulo, Sao Paulo, SP (Brazil)]. E-mail: anapaulagconverso@gmail.com; hfandrad@usp.br; Vieira, Daniel P. [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil); Instituto de Medicina Tropical de Sao Paulo, Sao Paulo, SP (Brazil); E-mail: dperezv@usp.br

2007-07-01

The improvement of techniques to detect pathogen agents in blood had reduced significantly the contamination mechanisms by hemocomponents in blood transfusion procedures. Ionizing radiation is a method that has presented several applications on medicine and in currently days has been showing special attention on blood banks which has been applied to avoid TA-GVHD development. DHFR is an enzyme constitutive in Plasmodium protozoa and has an important role in folate metabolism on these parasites. Detecting the expression of RNAm coder for this enzyme is possible to evaluate the viability of this parasite in blood samples. Plasmodium chabaudi AJ is a parasite that induces lethal malaria in rodents similar to human malaria In this work, the objective was to detect the presence of plasmodium protozoa in irradiated blood samples, infected experimentally, through the application of a RT-PCR using primers for the coder sequence of DHFR's mRNA. We studied doses of ionizing radiation between 0 and 75 Gy. The irradiation procedures were accomplished in Center of Radiation Technology of IPEN-CNEN in a {sup 60}Co panoramic source. Our results had demonstrated that RT-PCR is a sensible method to evaluate the viability of plasmodium in blood samples because the technique could detect low parasite burden in all tested samples. (author)

Establishment and in-house validation of stem-loop RT PCR method for MicroRNA398 expression analysis

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Timotijević Gordana S.

2015-01-01

Full Text Available MicroRNAs (miRNAs belong to the class of small non-coding RNAs which have important roles throughout development as well as in plant response to diverse environmental stresses. Some of plant miRNAs are essential for regulation and maintenance of nutritive homeostasis when nutrients are in excess or shortage comparing to optimal concentration for certain plant species. Better understanding of miRNAs functions implies development of efficient technology for profiling their gene expression. We set out to establish validate the methodology for miRNA gene expression analysis in cucumber grown under suboptimal mineral nutrient regimes, including iron deficiency. Reverse transcription by “stem-loop” primers in combination with Real time PCR method is one of potential approaches for quantification of miRNA gene expression. In this paper we presented a method for “stem loop” primer design specific for miR398, as well as reaction optimization and determination of Real time PCR efficiency. Proving the accuracy of this method was imperative as “stem loop” RT which consider separate transcription of target and endogenous control. The method was verified by comparison of the obtained data with results of miR398 expression achieved using a commercial kit based on simultaneous conversion of all RNAs in cDNAs. [Projekat Ministarstva nauke Republike Srbije, br. 173005 i br. ON-173028

Selection and Verification of Candidate Reference Genes for Mature MicroRNA Expression by Quantitative RT-PCR in the Tea Plant (Camellia sinensis

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Hui Song

2016-05-01

Full Text Available Quantitative reverse transcription-polymerase chain reaction (qRT-PCR is a rapid and sensitive method for analyzing microRNA (miRNA expression. However, accurate qRT-PCR results depend on the selection of reliable reference genes as internal positive controls. To date, few studies have identified reliable reference genes for differential expression analysis of miRNAs among tissues, and among experimental conditions in plants. In this study, three miRNAs and four non-coding small RNAs (ncRNA were selected as reference candidates, and the stability of their expression was evaluated among different tissues and under different experimental conditions in the tea plant (Camellia sinensis using the geNorm and NormFinder programs. It was shown that miR159a was the best single reference gene in the bud to the fifth leaf, 5S rRNA was the most suitable gene in different organs, miR6149 was the most stable gene when the leaves were attacked by Ectropis oblique and U4, miR5368n and miR159a were the best genes when the leaves were treated by methyl jasmonate (MeJA, salicylic acid (SA and abscisic acid (ABA, respectively. Our results provide suitable reference genes for future investigations on miRNA functions in tea plants.

Evaluation of reference genes for real-time quantitative PCR studies in Candida glabrata following azole treatment

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Li Qingdi

2012-06-01

Full Text Available Abstract Background The selection of stable and suitable reference genes for real-time quantitative PCR (RT-qPCR is a crucial prerequisite for reliable gene expression analysis under different experimental conditions. The present study aimed to identify reference genes as internal controls for gene expression studies by RT-qPCR in azole-stimulated Candida glabrata. Results The expression stability of 16 reference genes under fluconazole stress was evaluated using fold change and standard deviation computations with the hkgFinder tool. Our data revealed that the mRNA expression levels of three ribosomal RNAs (RDN5.8, RDN18, and RDN25 remained stable in response to fluconazole, while PGK1, UBC7, and UBC13 mRNAs showed only approximately 2.9-, 3.0-, and 2.5-fold induction by azole, respectively. By contrast, mRNA levels of the other 10 reference genes (ACT1, EF1α, GAPDH, PPIA, RPL2A, RPL10, RPL13A, SDHA, TUB1, and UBC4 were dramatically increased in C. glabrata following antifungal treatment, exhibiting changes ranging from 4.5- to 32.7-fold. We also assessed the expression stability of these reference genes using the 2-ΔΔCT method and three other software packages. The stability rankings of the reference genes by geNorm and the 2-ΔΔCT method were identical to those by hkgFinder, whereas the stability rankings by BestKeeper and NormFinder were notably different. We then validated the suitability of six candidate reference genes (ACT1, PGK1, RDN5.8, RDN18, UBC7, and UBC13 as internal controls for ten target genes in this system using the comparative CT method. Our validation experiments passed for all six reference genes analyzed except RDN18, where the amplification efficiency of RDN18 was different from that of the ten target genes. Finally, we demonstrated that the relative quantification of target gene expression varied according to the endogenous control used, highlighting the importance of the choice of internal controls in such

Real-time PCR in virology.

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Mackay, Ian M; Arden, Katherine E; Nitsche, Andreas

2002-03-15

The use of the polymerase chain reaction (PCR) in molecular diagnostics has increased to the point where it is now accepted as the gold standard for detecting nucleic acids from a number of origins and it has become an essential tool in the research laboratory. Real-time PCR has engendered wider acceptance of the PCR due to its improved rapidity, sensitivity, reproducibility and the reduced risk of carry-over contamination. There are currently five main chemistries used for the detection of PCR product during real-time PCR. These are the DNA binding fluorophores, the 5' endonuclease, adjacent linear and hairpin oligoprobes and the self-fluorescing amplicons, which are described in detail. We also discuss factors that have restricted the development of multiplex real-time PCR as well as the role of real-time PCR in quantitating nucleic acids. Both amplification hardware and the fluorogenic detection chemistries have evolved rapidly as the understanding of real-time PCR has developed and this review aims to update the scientist on the current state of the art. We describe the background, advantages and limitations of real-time PCR and we review the literature as it applies to virus detection in the routine and research laboratory in order to focus on one of the many areas in which the application of real-time PCR has provided significant methodological benefits and improved patient outcomes. However, the technology discussed has been applied to other areas of microbiology as well as studies of gene expression and genetic disease.

Evaluation of expression of NorA efflux pump in ciprofloxacin resistant Staphylococcus aureus against hexahydroquinoline derivative by real-time PCR.

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Pourmand, Mohammad Reza; Yousefi, Masoud; Salami, Seyed Alireza; Amini, Mohsen

2014-01-01

Staphylococcus aureus causes a wide variety of infections worldwide. Methicillin-resistant S. aureus is one of most common causes of nosocomial and community acquired infections. The fluoroquinolones are an important class of antibiotics that used to treat infections caused by S. aureus. Today, a significant increase in the rate of ciprofloxacin resistance in methicillin-resistant S. aureus strains is concerning. The norA efflux pump is considered as contributors to antibiotic resistance. Here, we aimed to evaluate the expression of norA efflux pump in the presence of hexahydroquinoline derivative in methicillin and ciprofloxacin resistant S. aureus. We were determined minimum inhibitory concentration of ciprofloxacin and hexahydroquinoline derivative and their combination by broth microdilution method against ciprofloxacin resistant S. aureus. The expression of the norA efflux pump gene was evaluated by quantitative Real-time PCR. This study showed that minimum inhibitory concentrations of ciprofloxacin in the presence of hexahydroquinoline derivative in comparison to ciprofloxacin were decreased. Quantitative Real-time PCR identified the increased expression of norA efflux pump gene in methicillin and ciprofloxacin resistant S. aureus strain. The increased expression of norA efflux pump gene may have resulted in the effort of S. aureus to survive. The results showed that the hexahydroquinoline derivative enhanced the antibacterial effect of ciprofloxacin against methicillin and ciprofloxacin resistant S. aureus. Therefore, the derivatives may be used as inhibitors of antibiotic resistance for combination therapy.

A longitudinal study of gene expression in healthy individuals

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Tessier Michel

2009-06-01

Full Text Available Abstract Background The use of gene expression in venous blood either as a pharmacodynamic marker in clinical trials of drugs or as a diagnostic test requires knowledge of the variability in expression over time in healthy volunteers. Here we defined a normal range of gene expression over 6 months in the blood of four cohorts of healthy men and women who were stratified by age (22–55 years and > 55 years and gender. Methods Eleven immunomodulatory genes likely to play important roles in inflammatory conditions such as rheumatoid arthritis and infection in addition to four genes typically used as reference genes were examined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR, as well as the full genome as represented by Affymetrix HG U133 Plus 2.0 microarrays. Results Gene expression levels as assessed by qRT-PCR and microarray were relatively stable over time with ~2% of genes as measured by microarray showing intra-subject differences over time periods longer than one month. Fifteen genes varied by gender. The eleven genes examined by qRT-PCR remained within a limited dynamic range for all individuals. Specifically, for the seven most stably expressed genes (CXCL1, HMOX1, IL1RN, IL1B, IL6R, PTGS2, and TNF, 95% of all samples profiled fell within 1.5–2.5 Ct, the equivalent of a 4- to 6-fold dynamic range. Two subjects who experienced severe adverse events of cancer and anemia, had microarray gene expression profiles that were distinct from normal while subjects who experienced an infection had only slightly elevated levels of inflammatory markers. Conclusion This study defines the range and variability of gene expression in healthy men and women over a six-month period. These parameters can be used to estimate the number of subjects needed to observe significant differences from normal gene expression in clinical studies. A set of genes that varied by gender was also identified as were a set of genes with elevated

Selection and evaluation of reference genes for RT-qPCR expression studies on Burkholderia tropica strain Ppe8, a sugarcane-associated diazotrophic bacterium grown with different carbon sources or sugarcane juice.

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da Silva, Paula Renata Alves; Vidal, Marcia Soares; de Paula Soares, Cleiton; Polese, Valéria; Simões-Araújo, Jean Luís; Baldani, José Ivo

2016-11-01

Among the members of the genus Burkholderia, Burkholderia tropica has the ability to fix nitrogen and promote sugarcane plant growth as well as act as a biological control agent. There is little information about how this bacterium metabolizes carbohydrates as well as those carbon sources found in the sugarcane juice that accumulates in stems during plant growth. Reverse transcription quantitative PCR (RT-qPCR) can be used to evaluate changes in gene expression during bacterial growth on different carbon sources. Here we tested the expression of six reference genes, lpxC, gyrB, recA, rpoA, rpoB, and rpoD, when cells were grown with glucose, fructose, sucrose, mannitol, aconitic acid, and sugarcane juice as carbon sources. The lpxC, gyrB, and recA were selected as the most stable reference genes based on geNorm and NormFinder software analyses. Validation of these three reference genes during strain Ppe8 growth on the same carbon sources showed that genes involved in glycogen biosynthesis (glgA, glgB, glgC) and trehalose biosynthesis (treY and treZ) were highly expressed when Ppe8 was grown in aconitic acid relative to other carbon sources, while otsA expression (trehalose biosynthesis) was reduced with all carbon sources. In addition, the expression level of the ORF_6066 (gluconolactonase) gene was reduced on sugarcane juice. The results confirmed the stability of the three selected reference genes (lpxC, gyrB, and recA) during the RT-qPCR and also their robustness by evaluating the relative expression of genes involved in glycogen and trehalose biosynthesis when strain Ppe8 was grown on different carbon sources and sugarcane juice.

Biomarker discovery for colon cancer using a 761 gene RT-PCR assay

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Hackett James R

2007-08-01

Full Text Available Abstract Background Reverse transcription PCR (RT-PCR is widely recognized to be the gold standard method for quantifying gene expression. Studies using RT-PCR technology as a discovery tool have historically been limited to relatively small gene sets compared to other gene expression platforms such as microarrays. We have recently shown that TaqMan® RT-PCR can be scaled up to profile expression for 192 genes in fixed paraffin-embedded (FPE clinical study tumor specimens. This technology has also been used to develop and commercialize a widely used clinical test for breast cancer prognosis and prediction, the Onco typeDX™ assay. A similar need exists in colon cancer for a test that provides information on the likelihood of disease recurrence in colon cancer (prognosis and the likelihood of tumor response to standard chemotherapy regimens (prediction. We have now scaled our RT-PCR assay to efficiently screen 761 biomarkers across hundreds of patient samples and applied this process to biomarker discovery in colon cancer. This screening strategy remains attractive due to the inherent advantages of maintaining platform consistency from discovery through clinical application. Results RNA was extracted from formalin fixed paraffin embedded (FPE tissue, as old as 28 years, from 354 patients enrolled in NSABP C-01 and C-02 colon cancer studies. Multiplexed reverse transcription reactions were performed using a gene specific primer pool containing 761 unique primers. PCR was performed as independent TaqMan® reactions for each candidate gene. Hierarchal clustering demonstrates that genes expected to co-express form obvious, distinct and in certain cases very tightly correlated clusters, validating the reliability of this technical approach to biomarker discovery. Conclusion We have developed a high throughput, quantitatively precise multi-analyte gene expression platform for biomarker discovery that approaches low density DNA arrays in numbers of

Selection of reference genes for RT-qPCR analysis in the monarch butterfly, Danaus plexippus (L.), a migrating bio-indicator

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Quantitative real-time PCR (qRT-PCR) is a reliable and reproducible technique for measuring and evaluating changes in gene expression. To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable housekeeping genes is required. In this study, expres...

Environmental regulation of plant gene expression: an RT-qPCR laboratory project for an upper-level undergraduate biochemistry or molecular biology course.

Science.gov (United States)

Eickelberg, Garrett J; Fisher, Alison J

2013-01-01

We present a novel laboratory project employing "real-time" RT-qPCR to measure the effect of environment on the expression of the FLOWERING LOCUS C gene, a key regulator of floral timing in Arabidopsis thaliana plants. The project requires four 3-hr laboratory sessions and is aimed at upper-level undergraduate students in biochemistry or molecular biology courses. The project provides students with hands-on experience with RT-qPCR, the current "gold standard" for gene expression analysis, including detailed data analysis using the common 2-ΔΔCT method. Moreover, it provides a convenient starting point for many inquiry-driven projects addressing diverse questions concerning ecological biochemistry, naturally occurring genetic variation, developmental biology, and the regulation of gene expression in nature. Copyright © 2013 Wiley Periodicals, Inc.

Exploring Valid Reference Genes for Quantitative Real-Time PCR Analysis in Sesamia inferens (Lepidoptera: Noctuidae)

OpenAIRE

Sun, Meng; Lu, Ming-Xing; Tang, Xiao-Tian; Du, Yu-Zhou

2015-01-01

The pink stem borer, Sesamia inferens, which is endemic in China and other parts of Asia, is a major pest of rice and causes significant yield loss in this host plant. Very few studies have addressed gene expression in S. inferens. Quantitative real-time PCR (qRT-PCR) is currently the most accurate and sensitive method for gene expression analysis. In qRT-PCR, data are normalized using reference genes, which help control for internal differences and reduce error between samples. In this study...

A probe-based qRT-PCR method to profile immunological gene expression in blood of captive beluga whales (Delphinapterus leucas

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Ming-An Tsai

2017-09-01

Full Text Available Cytokines are fundamental for a functioning immune system, and thus potentially serve as important indicators of animal health. Quantitation of mRNA using quantitative reverse transcription polymerase chain reaction (qRT-PCR is an established immunological technique. It is particularly suitable for detecting the expression of proteins against which monoclonal antibodies are not available. In this study, we developed a probe-based quantitative gene expression assay for immunological assessment of captive beluga whales (Delphinapterus leucas that is one of the most common cetacean species on display in aquariums worldwide. Six immunologically relevant genes (IL-2Rα, -4, -10, -12, TNFα, and IFNγ were selected for analysis, and two validated housekeeping genes (PGK1 and RPL4 with stable expression were used as reference genes. Sixteen blood samples were obtained from four animals with different health conditions and stored in RNAlater™ solution. These samples were used for RNA extraction followed by qRT-PCR analysis. Analysis of gene transcripts was performed by relative quantitation using the comparative Cq method with the integration of amplification efficiency and two reference genes. The expression levels of each gene in the samples from clinically healthy animals were normally distributed. Transcript outliers for IL-2Rα, IL-4, IL-12, TNFα, and IFNγ were noticed in four samples collected from two clinically unhealthy animals. This assay has the potential to identify immune system deviation from normal state, which is caused by health problems. Furthermore, knowing the immune status of captive cetaceans could help both trainers and veterinarians in implementing preventive approaches prior to disease onset.

Clinical utility of RT-PCR in assessing HER 2 gene expression versus traditional IHC and FISH in breast cancer patients.

Science.gov (United States)

Suryavanshi, Moushumi; Mehta, Anurag; Jaipuria, Jiten; Kumar, Dushyant; Vishwakarma, Gayatri; Panigrahi, Manoj Kumar; Verma, Haristuti; Saifi, Mumtaz; Sharma, Sanjeev; Tandon, Simran; Doval, D C; Das, Bhudev C

2018-02-09

IHC and FISH are used for categorizing HER 2 status in breast cancer at the protein and DNA level, respectively. HER 2 expression at the RNA level is quantitative, cheaper, easier to standardize and free from interobserver variation. 115 consecutive patients were tested by IHC, FISH and RT-PCR (test cohort). Assuming FISH result to be the response variable, ROC curves for RT-PCR ratio were analyzed to label HER 2 negative, equivocal and positive cases as RT-PCR score 1, 2 and 3, respectively. Inter-relationships between RT-PCR, IHC and FISH were defined. 'Clinical benefit' of a test was defined as proportion of patients labeled unequivocally as HER 2 positive or negative. Population for 1 year was simulated constraint to previous reports of HER 2 positivity and IHC category distribution by a meta-analysis of previous studies that evaluated concordance between IHC and FISH to determine HER 2 status (simulation cohort). Four diagnostic pathways in the simulation cohort were defined-(1) initial IHC, followed by FISH (conventional pathway); (2) initial RT-PCR, followed by FISH; (3) initial IHC, followed by RT-PCR and then by FISH; (4) initial RT-PCR, followed by IHC and then by FISH. The clinical benefit of IHC and RT-PCR in the four pathways was analyzed and sensitivity analysis for incremental cost-effectiveness ratio and cost-benefit comapring RT-PCR against IHC, both as first-line tests and among those with IHC score 2 as a reflex second-line test was performed by the Monte Carlo technique. 115 patients comprised the study population. While none with IHC score of 0 or 1 was FISH positive for HER 2, all cases with IHC score of 3 were FISH positive. 43 cases were assigned IHC score of 2. Thus, 72 patients benefited from the initial IHC testing [clinical benefit 62.6%], with the overall concordance between IHC and FISH being 100% for those with IHC score of 0, 1 and 3 (conclusive IHC categories). For RT-PCR with 100% concordance, 15.7% (115-97 = 18) patients

Genome-Wide Identification and Evaluation of Reference Genes for Quantitative RT-PCR Analysis during Tomato Fruit Development.

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Cheng, Yuan; Bian, Wuying; Pang, Xin; Yu, Jiahong; Ahammed, Golam J; Zhou, Guozhi; Wang, Rongqing; Ruan, Meiying; Li, Zhimiao; Ye, Qingjing; Yao, Zhuping; Yang, Yuejian; Wan, Hongjian

2017-01-01

Gene expression analysis in tomato fruit has drawn increasing attention nowadays. Quantitative real-time PCR (qPCR) is a routine technique for gene expression analysis. In qPCR operation, reliability of results largely depends on the choice of appropriate reference genes (RGs). Although tomato is a model for fruit biology study, few RGs for qPCR analysis in tomato fruit had yet been developed. In this study, we initially identified 38 most stably expressed genes based on tomato transcriptome data set, and their expression stabilities were further determined in a set of tomato fruit samples of four different fruit developmental stages (Immature, mature green, breaker, mature red) using qPCR analysis. Two statistical algorithms, geNorm and Normfinder, concordantly determined the superiority of these identified putative RGs. Notably, SlFRG05 (Solyc01g104170), SlFRG12 (Solyc04g009770), SlFRG16 (Solyc10g081190), SlFRG27 (Solyc06g007510), and SlFRG37 (Solyc11g005330) were proved to be suitable RGs for tomato fruit development study. Further analysis using geNorm indicate that the combined use of SlFRG03 (Solyc02g063070) and SlFRG27 would provide more reliable normalization results in qPCR experiments. The identified RGs in this study will be beneficial for future qPCR analysis of tomato fruit developmental study, as well as for the potential identification of optimal normalization controls in other plant species.

Genome-Wide Identification and Evaluation of Reference Genes for Quantitative RT-PCR Analysis during Tomato Fruit Development

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Yuan Cheng

2017-08-01

Full Text Available Gene expression analysis in tomato fruit has drawn increasing attention nowadays. Quantitative real-time PCR (qPCR is a routine technique for gene expression analysis. In qPCR operation, reliability of results largely depends on the choice of appropriate reference genes (RGs. Although tomato is a model for fruit biology study, few RGs for qPCR analysis in tomato fruit had yet been developed. In this study, we initially identified 38 most stably expressed genes based on tomato transcriptome data set, and their expression stabilities were further determined in a set of tomato fruit samples of four different fruit developmental stages (Immature, mature green, breaker, mature red using qPCR analysis. Two statistical algorithms, geNorm and Normfinder, concordantly determined the superiority of these identified putative RGs. Notably, SlFRG05 (Solyc01g104170, SlFRG12 (Solyc04g009770, SlFRG16 (Solyc10g081190, SlFRG27 (Solyc06g007510, and SlFRG37 (Solyc11g005330 were proved to be suitable RGs for tomato fruit development study. Further analysis using geNorm indicate that the combined use of SlFRG03 (Solyc02g063070 and SlFRG27 would provide more reliable normalization results in qPCR experiments. The identified RGs in this study will be beneficial for future qPCR analysis of tomato fruit developmental study, as well as for the potential identification of optimal normalization controls in other plant species.

Construction of an adult barnacle (Balanus amphitrite cDNA library and selection of reference genes for quantitative RT-PCR studies

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Burgess J Grant

2009-06-01

Full Text Available Abstract Background Balanus amphitrite is a barnacle commonly used in biofouling research. Although many aspects of its biology have been elucidated, the lack of genetic information is impeding a molecular understanding of its life cycle. As part of a wider multidisciplinary approach to reveal the biogenic cues influencing barnacle settlement and metamorphosis, we have sequenced and annotated the first cDNA library for B. amphitrite. We also present a systematic validation of potential reference genes for normalization of quantitative real-time PCR (qRT-PCR data obtained from different developmental stages of this animal. Results We generated a cDNA library containing expressed sequence tags (ESTs from adult B. amphitrite. A total of 609 unique sequences (comprising 79 assembled clusters and 530 singlets were derived from 905 reliable unidirectionally sequenced ESTs. Bioinformatics tools such as BLAST, HMMer and InterPro were employed to allow functional annotation of the ESTs. Based on these analyses, we selected 11 genes to study their ability to normalize qRT-PCR data. Total RNA extracted from 7 developmental stages was reverse transcribed and the expression stability of the selected genes was compared using geNorm, BestKeeper and NormFinder. These software programs produced highly comparable results, with the most stable gene being mt-cyb, while tuba, tubb and cp1 were clearly unsuitable for data normalization. Conclusion The collection of B. amphitrite ESTs and their annotation has been made publically available representing an important resource for both basic and applied research on this species. We developed a qRT-PCR assay to determine the most reliable reference genes. Transcripts encoding cytochrome b and NADH dehydrogenase subunit 1 were expressed most stably, although other genes also performed well and could prove useful to normalize gene expression studies.

Selection of reference genes for quantitative RT-PCR (RT-qPCR) analysis of rat tissues under physiological and toxicological conditions

DEFF Research Database (Denmark)

Svingen, Terje; Letting, Heidi; Hadrup, Niels

2015-01-01

In biological research the analysis of gene expression levels in cells and tissues can be a powerful tool to gain insights into biological processes. For this, quantitative RT-PCR (RT-qPCR) is a popular method that often involve the use of constitutively expressed endogenous reference (or...... ‘housekeeping’) gene for normalization of data. Thus, it is essential to use reference genes that have been verified to be stably expressed within the specific experimental setting. Here, we have analysed the expression stability of 12 commonly used reference genes (Actb, B2m, Gapdh, Hprt, Pgk1, Rn18s, Rpl13a...

Selection of reliable reference genes for RT-qPCR studies in Octopus vulgaris paralarvae during development and immune-stimulation.

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García-Fernández, P; Castellanos-Martínez, S; Iglesias, J; Otero, J J; Gestal, C

2016-07-01

The common octopus, Octopus vulgaris is a new candidate species for aquaculture. However, rearing of octopus paralarvae is hampered by high mortality and poor growth rates that impede its entire culture. The study of genes involved in the octopus development and immune response capability could help to understand the key of paralarvae survival and thus, to complete the octopus life cycle. Quantitative real-time PCR (RT-qPCR) is the most frequently tool used to quantify the gene expression because of specificity and sensitivity. However, reliability of RT-qPCR requires the selection of appropriate normalization genes whose expression must be stable across the different experimental conditions of the study. Hence, the aim of the present work is to evaluate the stability of six candidate genes: β-actin (ACT), elongation factor 1-α (EF), ubiquitin (UBI), β-tubulin (TUB), glyceraldehyde 3-phosphate dehydrogenase (GADPH) and ribosomal RNA 18 (18S) in order to select the best reference gene. The stability of gene expression was analyzed using geNorm, NormFinder and Bestkeeper, in octopus paralarvae of seven developmental stages (embryo, paralarvae of 0, 10, 15, 20, 30 and 34days) and paralarvae of 20days after challenge with Vibrio lentus and Vibrio splendidus. The results were validated by measuring the expression of PGRP, a stimuli-specific gene. Our results showed UBI, EF and 18S as the most suitable reference genes during development of octopus paralarvae, and UBI, ACT and 18S for bacterial infection. These results provide a basis for further studies exploring molecular mechanism of their development and innate immune defense. Copyright © 2016 Elsevier Inc. All rights reserved.

Selection of appropriate reference genes for the detection of rhythmic gene expression via quantitative real-time PCR in Tibetan hulless barley.

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Jing Cai

Full Text Available Hulless barley (Hordeum vulgare L. var. nudum. hook. f. has been cultivated as a major crop in the Qinghai-Tibet plateau of China for thousands of years. Compared to other cereal crops, the Tibetan hulless barley has developed stronger endogenous resistances to survive in the severe environment of its habitat. To understand the unique resistant mechanisms of this plant, detailed genetic studies need to be performed. The quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR is the most commonly used method in detecting gene expression. However, the selection of stable reference genes under limited experimental conditions was considered to be an essential step for obtaining accurate results in qRT-PCR. In this study, 10 candidate reference genes-ACT (Actin, E2 (Ubiquitin conjugating enzyme 2, TUBα (Alpha-tubulin, TUBβ6 (Beta-tubulin 6, GAPDH (Glyceraldehyde 3-phosphate dehydrogenase, EF-1α (Elongation factor 1-alpha, SAMDC (S-adenosylmethionine decarboxylase, PKABA1 (Gene for protein kinase HvPKABA1, PGK (Phosphoglycerate kinase, and HSP90 (Heat shock protein 90-were selected from the NCBI gene database of barley. Following qRT-PCR amplifications of all candidate reference genes in Tibetan hulless barley seedlings under various stressed conditions, the stabilities of these candidates were analyzed by three individual software packages including geNorm, NormFinder, and BestKeeper. The results demonstrated that TUBβ6, E2, TUBα, and HSP90 were generally the most suitable sets under all tested conditions; similarly, TUBα and HSP90 showed peak stability under salt stress, TUBα and EF-1α were the most suitable reference genes under cold stress, and ACT and E2 were the most stable under drought stress. Finally, a known circadian gene CCA1 was used to verify the service ability of chosen reference genes. The results confirmed that all recommended reference genes by the three software were suitable for gene expression

Selection of appropriate reference genes for the detection of rhythmic gene expression via quantitative real-time PCR in Tibetan hulless barley.

Science.gov (United States)

Cai, Jing; Li, Pengfei; Luo, Xiao; Chang, Tianliang; Li, Jiaxing; Zhao, Yuwei; Xu, Yao

2018-01-01

Hulless barley (Hordeum vulgare L. var. nudum. hook. f.) has been cultivated as a major crop in the Qinghai-Tibet plateau of China for thousands of years. Compared to other cereal crops, the Tibetan hulless barley has developed stronger endogenous resistances to survive in the severe environment of its habitat. To understand the unique resistant mechanisms of this plant, detailed genetic studies need to be performed. The quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is the most commonly used method in detecting gene expression. However, the selection of stable reference genes under limited experimental conditions was considered to be an essential step for obtaining accurate results in qRT-PCR. In this study, 10 candidate reference genes-ACT (Actin), E2 (Ubiquitin conjugating enzyme 2), TUBα (Alpha-tubulin), TUBβ6 (Beta-tubulin 6), GAPDH (Glyceraldehyde 3-phosphate dehydrogenase), EF-1α (Elongation factor 1-alpha), SAMDC (S-adenosylmethionine decarboxylase), PKABA1 (Gene for protein kinase HvPKABA1), PGK (Phosphoglycerate kinase), and HSP90 (Heat shock protein 90)-were selected from the NCBI gene database of barley. Following qRT-PCR amplifications of all candidate reference genes in Tibetan hulless barley seedlings under various stressed conditions, the stabilities of these candidates were analyzed by three individual software packages including geNorm, NormFinder, and BestKeeper. The results demonstrated that TUBβ6, E2, TUBα, and HSP90 were generally the most suitable sets under all tested conditions; similarly, TUBα and HSP90 showed peak stability under salt stress, TUBα and EF-1α were the most suitable reference genes under cold stress, and ACT and E2 were the most stable under drought stress. Finally, a known circadian gene CCA1 was used to verify the service ability of chosen reference genes. The results confirmed that all recommended reference genes by the three software were suitable for gene expression analysis

Identification of Reference Genes for Normalizing Quantitative Real-Time PCR in Urechis unicinctus

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Bai, Yajiao; Zhou, Di; Wei, Maokai; Xie, Yueyang; Gao, Beibei; Qin, Zhenkui; Zhang, Zhifeng

2018-06-01

The reverse transcription quantitative real-time PCR (RT-qPCR) has become one of the most important techniques of studying gene expression. A set of valid reference genes are essential for the accurate normalization of data. In this study, five candidate genes were analyzed with geNorm, NormFinder, BestKeeper and ΔCt methods to identify the genes stably expressed in echiuran Urechis unicinctus, an important commercial marine benthic worm, under abiotic (sulfide stress) and normal (adult tissues, embryos and larvae at different development stages) conditions. The comprehensive results indicated that the expression of TBP was the most stable at sulfide stress and in developmental process, while the expression of EF- 1- α was the most stable at sulfide stress and in various tissues. TBP and EF- 1- α were recommended as a suitable reference gene combination to accurately normalize the expression of target genes at sulfide stress; and EF- 1- α, TBP and TUB were considered as a potential reference gene combination for normalizing the expression of target genes in different tissues. No suitable gene combination was obtained among these five candidate genes for normalizing the expression of target genes for developmental process of U. unicinctus. Our results provided a valuable support for quantifying gene expression using RT-qPCR in U. unicinctus.

Evaluation of suitable reference genes for gene expression studies ...

Indian Academy of Sciences (India)

2011-12-14

Dec 14, 2011 ... MADS family of TFs control floral organ identity within each whorl of the flower by activating downstream genes. Measuring gene expression in different tissue types and developmental stages is of fundamental importance in TFs functional research. In last few years, quantitative real-time. PCR (qRT-PCR) ...

Identification and comprehensive evaluation of reference genes for RT-qPCR analysis of host gene-expression in Brassica juncea-aphid interaction using microarray data.

Science.gov (United States)

Ram, Chet; Koramutla, Murali Krishna; Bhattacharya, Ramcharan

2017-07-01

Brassica juncea is a chief oil yielding crop in many parts of the world including India. With advancement of molecular techniques, RT-qPCR based study of gene-expression has become an integral part of experimentations in crop breeding. In RT-qPCR, use of appropriate reference gene(s) is pivotal. The virtue of the reference genes, being constant in expression throughout the experimental treatments, needs to be validated case by case. Appropriate reference gene(s) for normalization of gene-expression data in B. juncea during the biotic stress of aphid infestation is not known. In the present investigation, 11 reference genes identified from microarray database of Arabidopsis-aphid interaction at a cut off FDR ≤0.1, along with two known reference genes of B. juncea, were analyzed for their expression stability upon aphid infestation. These included 6 frequently used and 5 newly identified reference genes. Ranking orders of the reference genes in terms of expression stability were calculated using advanced statistical approaches such as geNorm, NormFinder, delta Ct and BestKeeper. The analysis suggested CAC, TUA and DUF179 as the most suitable reference genes. Further, normalization of the gene-expression data of STP4 and PR1 by the most and the least stable reference gene, respectively has demonstrated importance and applicability of the recommended reference genes in aphid infested samples of B. juncea. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Identification and validation of superior reference gene for gene expression normalization via RT-qPCR in staminate and pistillate flowers of Jatropha curcas - A biodiesel plant.

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Karuppaiya, Palaniyandi; Yan, Xiao-Xue; Liao, Wang; Wu, Jun; Chen, Fang; Tang, Lin

2017-01-01

Physic nut (Jatropha curcas L) seed oil is a natural resource for the alternative production of fossil fuel. Seed oil production is mainly depended on seed yield, which was restricted by the low ratio of staminate flowers to pistillate flowers. Further, the mechanism of physic nut flower sex differentiation has not been fully understood yet. Quantitative Real Time-Polymerase Chain Reaction is a reliable and widely used technique to quantify the gene expression pattern in biological samples. However, for accuracy of qRT-PCR, appropriate reference gene is highly desirable to quantify the target gene level. Hence, the present study was aimed to identify the stable reference genes in staminate and pistillate flowers of J. curcas. In this study, 10 candidate reference genes were selected and evaluated for their expression stability in staminate and pistillate flowers, and their stability was validated by five different algorithms (ΔCt, BestKeeper, NormFinder, GeNorm and RefFinder). Resulting, TUB and EF found to be the two most stably expressed reference for staminate flower; while GAPDH1 and EF found to be the most stably expressed reference gene for pistillate flowers. Finally, RT-qPCR assays of target gene AGAMOUS using the identified most stable reference genes confirmed the reliability of selected reference genes in different stages of flower development. AGAMOUS gene expression levels at different stages were further proved by gene copy number analysis. Therefore, the present study provides guidance for selecting appropriate reference genes for analyzing the expression pattern of floral developmental genes in staminate and pistillate flowers of J. curcas.

Identification of valid reference genes for the normalization of RT-qPCR expression studies in human breast cancer cell lines treated with and without transient transfection.

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Lin-Lin Liu

Full Text Available Reverse transcription-quantitative polymerase chain reaction (RT-qPCR is a powerful technique for examining gene expression changes during tumorigenesis. Target gene expression is generally normalized by a stably expressed endogenous reference gene; however, reference gene expression may differ among tissues under various circumstances. Because no valid reference genes have been documented for human breast cancer cell lines containing different cancer subtypes treated with transient transfection, we identified appropriate and reliable reference genes from thirteen candidates in a panel of 10 normal and cancerous human breast cell lines under experimental conditions with/without transfection treatments with two transfection reagents. Reference gene expression stability was calculated using four algorithms (geNorm, NormFinder, BestKeeper and comparative delta Ct, and the recommended comprehensive ranking was provided using geometric means of the ranking values using the RefFinder tool. GeNorm analysis revealed that two reference genes should be sufficient for all cases in this study. A stability analysis suggests that 18S rRNA-ACTB is the best reference gene combination across all cell lines; ACTB-GAPDH is best for basal breast cancer cell lines; and HSPCB-ACTB is best for ER+ breast cancer cells. After transfection, the stability ranking of the reference gene fluctuated, especially with Lipofectamine 2000 transfection reagent in two subtypes of basal and ER+ breast cell lines. Comparisons of relative target gene (HER2 expression revealed different expressional patterns depending on the reference genes used for normalization. We suggest that identifying the most stable and suitable reference genes is critical for studying specific cell lines under certain circumstances.

Unstable Expression of Commonly Used Reference Genes in Rat Pancreatic Islets Early after Isolation Affects Results of Gene Expression Studies.

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Lucie Kosinová

Full Text Available The use of RT-qPCR provides a powerful tool for gene expression studies; however, the proper interpretation of the obtained data is crucially dependent on accurate normalization based on stable reference genes. Recently, strong evidence has been shown indicating that the expression of many commonly used reference genes may vary significantly due to diverse experimental conditions. The isolation of pancreatic islets is a complicated procedure which creates severe mechanical and metabolic stress leading possibly to cellular damage and alteration of gene expression. Despite of this, freshly isolated islets frequently serve as a control in various gene expression and intervention studies. The aim of our study was to determine expression of 16 candidate reference genes and one gene of interest (F3 in isolated rat pancreatic islets during short-term cultivation in order to find a suitable endogenous control for gene expression studies. We compared the expression stability of the most commonly used reference genes and evaluated the reliability of relative and absolute quantification using RT-qPCR during 0-120 hrs after isolation. In freshly isolated islets, the expression of all tested genes was markedly depressed and it increased several times throughout the first 48 hrs of cultivation. We observed significant variability among samples at 0 and 24 hrs but substantial stabilization from 48 hrs onwards. During the first 48 hrs, relative quantification failed to reflect the real changes in respective mRNA concentrations while in the interval 48-120 hrs, the relative expression generally paralleled the results determined by absolute quantification. Thus, our data call into question the suitability of relative quantification for gene expression analysis in pancreatic islets during the first 48 hrs of cultivation, as the results may be significantly affected by unstable expression of reference genes. However, this method could provide reliable information

Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay

Science.gov (United States)

Ogrean, Christy; Jackson, Ben; Covino, James

2010-01-01

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and accuracy of the assay. These primer/probe sets are pre-designed to >98% of the human and mouse genomes and feature significant improvements from previously available technologies. These improvements were made possible by virtue of a novel design algorithm, developed by Thermo Scientific bioinformatics experts. Several convenient features have been incorporated into the Solaris qPCR Assay to streamline the process of performing quantitative real-time PCR. First, the protocol is similar to commonly employed alternatives, so the methods used during qPCR are likely to be familiar. Second, the master mix is blue, which makes setting the qPCR reactions easier to track. Third, the thermal cycling conditions are the same for all assays (genes), making it possible to run many samples at a time and reducing the potential for error. Finally, the probe and primer sequence information are provided, simplifying the publication process. Here, we demonstrate how to obtain the appropriate Solaris reagents using the GENEius product search feature found on the ordering web site (www.thermo.com/solaris) and how to use the Solaris reagents for performing qPCR using the standard curve method. PMID:20567213

Identification and validation of reference genes for qRT-PCR studies of the obligate aphid pathogenic fungus Pandora neoaphidis during different developmental stages.

Science.gov (United States)

Zhang, Shutao; Chen, Chun; Xie, Tingna; Ye, Sudan

2017-01-01

The selection of stable reference genes is a critical step for the accurate quantification of gene expression. To identify and validate the reference genes in Pandora neoaphidis-an obligate aphid pathogenic fungus-the expression of 13classical candidate reference genes were evaluated by quantitative real-time reverse transcriptase polymerase chain reaction(qPCR) at four developmental stages (conidia, conidia with germ tubes, short hyphae and elongated hyphae). Four statistical algorithms, including geNorm, NormFinder, BestKeeper and Delta Ct method were used to rank putative reference genes according to their expression stability and indicate the best reference gene or combination of reference genes for accurate normalization. The analysis of comprehensive ranking revealed that ACT1and 18Swas the most stably expressed genes throughout the developmental stages. To further validate the suitability of the reference genes identified in this study, the expression of cell division control protein 25 (CDC25) and Chitinase 1(CHI1) genes were used to further confirm the validated candidate reference genes. Our study presented the first systematic study of reference gene(s) selection for P. neoaphidis study and provided guidelines to obtain more accurate qPCR results for future developmental efforts.

Whole blood Nested PCR and Real-time PCR amplification of Talaromyces marneffei specific DNA for diagnosis.

Science.gov (United States)

Lu, Sha; Li, Xiqing; Calderone, Richard; Zhang, Jing; Ma, Jianchi; Cai, Wenying; Xi, Liyan

2016-02-01

Talaromyces marneffei is a dimorphic pathogenic fungus, which is a life-threatening invasive mycosis in the immunocompromised host. Prompt diagnosis of T. marneffei infection remains difficult although there has been progress in attempts to expedite the diagnosis of this infection. We previously demonstrated the value of nested polymerase chain reaction (PCR) to detect T. marneffei in paraffin embedded tissue samples with high sensitivity and specificity. In this study, this assay was used to detect the DNA of T. marneffei in whole blood samples. Real-time PCR assay was also evaluated to identify T. marneffei in the same samples. Twenty out of 30 whole blood samples (67%) collected from 23 patients were found positive by using the nested PCR assay, while 23/30 (77%) samples were found positive by using the real-time PCR assay. In order to express accurately the fungal loads, we used a normalized linearized plasmid as an internal control for real-time PCR. The assay results were correlated as the initial quantity (copies/μl) with fungal burden. These data indicate that combination of nested PCR and real-time PCR assay provides an attractive alternative for identification of T. marneffei DNA in whole blood samples of HIV-infected patients. © The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Design and optimization of reverse-transcription quantitative PCR experiments.

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Tichopad, Ales; Kitchen, Rob; Riedmaier, Irmgard; Becker, Christiane; Ståhlberg, Anders; Kubista, Mikael

2009-10-01

Quantitative PCR (qPCR) is a valuable technique for accurately and reliably profiling and quantifying gene expression. Typically, samples obtained from the organism of study have to be processed via several preparative steps before qPCR. We estimated the errors of sample withdrawal and extraction, reverse transcription (RT), and qPCR that are introduced into measurements of mRNA concentrations. We performed hierarchically arranged experiments with 3 animals, 3 samples, 3 RT reactions, and 3 qPCRs and quantified the expression of several genes in solid tissue, blood, cell culture, and single cells. A nested ANOVA design was used to model the experiments, and relative and absolute errors were calculated with this model for each processing level in the hierarchical design. We found that intersubject differences became easily confounded by sample heterogeneity for single cells and solid tissue. In cell cultures and blood, the noise from the RT and qPCR steps contributed substantially to the overall error because the sampling noise was less pronounced. We recommend the use of sample replicates preferentially to any other replicates when working with solid tissue, cell cultures, and single cells, and we recommend the use of RT replicates when working with blood. We show how an optimal sampling plan can be calculated for a limited budget. .

Regulation of bacterial photosynthesis genes by the small noncoding RNA PcrZ.

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Mank, Nils N; Berghoff, Bork A; Hermanns, Yannick N; Klug, Gabriele

2012-10-02

The small RNA PcrZ (photosynthesis control RNA Z) of the facultative phototrophic bacterium Rhodobacter sphaeroides is induced upon a drop of oxygen tension with similar kinetics to those of genes for components of photosynthetic complexes. High expression of PcrZ depends on PrrA, the response regulator of the PrrB/PrrA two-component system with a central role in redox regulation in R. sphaeroides. In addition the FnrL protein, an activator of some photosynthesis genes at low oxygen tension, is involved in redox-dependent expression of this small (s)RNA. Overexpression of full-length PcrZ in R. sphaeroides affects expression of a small subset of genes, most of them with a function in photosynthesis. Some mRNAs from the photosynthetic gene cluster were predicted to be putative PcrZ targets and results from an in vivo reporter system support these predictions. Our data reveal a negative effect of PcrZ on expression of its target mRNAs. Thus, PcrZ counteracts the redox-dependent induction of photosynthesis genes, which is mediated by protein regulators. Because PrrA directly activates photosynthesis genes and at the same time PcrZ, which negatively affects photosynthesis gene expression, this is one of the rare cases of an incoherent feed-forward loop including an sRNA. Our data identified PcrZ as a trans acting sRNA with a direct regulatory function in formation of photosynthetic complexes and provide a model for the control of photosynthesis gene expression by a regulatory network consisting of proteins and a small noncoding RNA.

Isolation of MA-ACS Gene Family and Expression Study of MA-ACS1 Gene in Musa acuminata Cultivar Pisang Ambon Lumut

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LISTYA UTAMI KARMAWAN

2009-03-01

Full Text Available Musa acuminata cultivar pisang ambon lumut is a native climacteric fruit from Indonesia. Climacteric fruit ripening process is triggered by the gaseous plant hormone ethylene. The rate limiting enzyme involved in ethylene biosynthesis is ACC synthase (ACS which is encoded by ACS gene family. The objective of this study is to identify MA-ACS gene family in M. acuminata cultivar pisang ambon lumut and to study the MA-ACS1 gene expression. The result showed that there were nine M. acuminata ACS gene family members called MA-ACS1–9. Two of them (MA-ACS1 and MA-ACS2 were assessed using reverse transcriptase PCR (RT-PCR for gene expression study and it was only MA-ACS1 correlated with fruit ripening. The MA-ACS1 gene fragment has been successfully isolated and characterized and it has three introns, four exons, and one stop codon. It also shows highest homology with MACS1 gene from M. acuminata cultivar Hsian Jien Chiao (GenBank accession number AF056164. Expression analysis of MA-ACS1 using quantitative PCR (qPCR showed that MA-ACS1 gene expression increased significantly in the third day, reached maximum at the fifth day, and then decreased in the seventh day after harvesting. The qPCR expression analysis result correlated with the result of physical analysis during fruit ripening.

Microgravity validation of a novel system for RNA isolation and multiplex quantitative real time PCR analysis of gene expression on the International Space Station.

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Macarena Parra

Full Text Available The International Space Station (ISS National Laboratory is dedicated to studying the effects of space on life and physical systems, and to developing new science and technologies for space exploration. A key aspect of achieving these goals is to operate the ISS National Lab more like an Earth-based laboratory, conducting complex end-to-end experimentation, not limited to simple microgravity exposure. Towards that end NASA developed a novel suite of molecular biology laboratory tools, reagents, and methods, named WetLab-2, uniquely designed to operate in microgravity, and to process biological samples for real-time gene expression analysis on-orbit. This includes a novel fluidic RNA Sample Preparation Module and fluid transfer devices, all-in-one lyophilized PCR assays, centrifuge, and a real-time PCR thermal cycler. Here we describe the results from the WetLab-2 validation experiments conducted in microgravity during ISS increment 47/SPX-8. Specifically, quantitative PCR was performed on a concentration series of DNA calibration standards, and Reverse Transcriptase-quantitative PCR was conducted on RNA extracted and purified on-orbit from frozen Escherichia coli and mouse liver tissue. Cycle threshold (Ct values and PCR efficiencies obtained on-orbit from DNA standards were similar to Earth (1 g controls. Also, on-orbit multiplex analysis of gene expression from bacterial cells and mammalian tissue RNA samples was successfully conducted in about 3 h, with data transmitted within 2 h of experiment completion. Thermal cycling in microgravity resulted in the trapping of gas bubbles inside septa cap assay tubes, causing small but measurable increases in Ct curve noise and variability. Bubble formation was successfully suppressed in a rapid follow-up on-orbit experiment using standard caps to pressurize PCR tubes and reduce gas release during heating cycles. The WetLab-2 facility now provides a novel operational on-orbit research capability for

Real-time quantitative PCR of Staphylococcus aureus and application in restaurant meals.

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Berrada, H; Soriano, J M; Mañes, J; Picó, Y

2006-01-01

Staphylococcus aureus is considered the second most common pathogen to cause outbreaks of food poisoning, exceeded only by Campylobacter. Consumption of foods containing this microorganism is often identified as the cause of illness. In this study, a rapid, reliable, and sensitive real-time quantitative PCR was developed and compared with conventional culture methods. Real-time quantitative PCR was carried out by purifying DNA extracts of S. aureus with a Staphylococcus sample preparation kit and quantifying it in the LightCycler system with hybridization probes. The assay was linear from a range of 10 to 10(6) S. aureus cells (r2 > 0.997). The PCR reaction presented an efficiency of >85%. Accuracy of the PCR-based assay, expressed as percent bias, was around 13%, and the precision, expressed as a percentage of the coefficient of variation, was 7 to 10%. Intraday and interday variability were studied at 10(2) CFU/g and was 12 and 14%, respectively. The proposed method was applied to the analysis of 77 samples of restaurant meals in Valencia (Spain). In 11.6% of samples S. aureus was detected by real-time quantitative PCR, as well as by the conventional microbiological method. An excellent correspondence between real-time quantitative PCR and microbiological numbers (CFU/g) was observed with deviations of < 28%.

Multiway real-time PCR gene expression profiling in yeast. Saccharomyces cerevisiae reveals altered transcriptional response of ADH-genes to glucose stimuli

Czech Academy of Sciences Publication Activity Database

Stahlberg, A.; Elbing, K.; Andrade-Garda, J.M.; Sjögreen, B.; Forootan, A.; Kubista, Mikael

2008-01-01

Roč. 9, č. 170 (2008), s. 1-41 ISSN 1471-2164 Institutional research plan: CEZ:AV0Z50520701 Keywords : Expression Profiling * Real-time PCR * Yeast Subject RIV: EI - Biotechnology ; Bionics Impact factor: 3.926, year: 2008

Transcriptome-wide selection of a reliable set of reference genes for gene expression studies in potato cyst nematodes (Globodera spp.).

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Sabeh, Michael; Duceppe, Marc-Olivier; St-Arnaud, Marc; Mimee, Benjamin

2018-01-01

Relative gene expression analyses by qRT-PCR (quantitative reverse transcription PCR) require an internal control to normalize the expression data of genes of interest and eliminate the unwanted variation introduced by sample preparation. A perfect reference gene should have a constant expression level under all the experimental conditions. However, the same few housekeeping genes selected from the literature or successfully used in previous unrelated experiments are often routinely used in new conditions without proper validation of their stability across treatments. The advent of RNA-Seq and the availability of public datasets for numerous organisms are opening the way to finding better reference genes for expression studies. Globodera rostochiensis is a plant-parasitic nematode that is particularly yield-limiting for potato. The aim of our study was to identify a reliable set of reference genes to study G. rostochiensis gene expression. Gene expression levels from an RNA-Seq database were used to identify putative reference genes and were validated with qRT-PCR analysis. Three genes, GR, PMP-3, and aaRS, were found to be very stable within the experimental conditions of this study and are proposed as reference genes for future work.

Optimization and analysis of a quantitative real-time PCR-based technique to determine microRNA expression in formalin-fixed paraffin-embedded samples

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Reis Patricia P

2010-06-01

Full Text Available Abstract Background MicroRNAs (miRs are non-coding RNA molecules involved in post-transcriptional regulation, with diverse functions in tissue development, differentiation, cell proliferation and apoptosis. miRs may be less prone to degradation during formalin fixation, facilitating miR expression studies in formalin-fixed paraffin-embedded (FFPE tissue. Results Our study demonstrates that the TaqMan Human MicroRNA Array v1.0 (Early Access platform is suitable for miR expression analysis in FFPE tissue with a high reproducibility (correlation coefficients of 0.95 between duplicates, p 35, we show that reproducibility between technical replicates, equivalent dilutions, and FFPE vs. frozen samples is best in the high abundance stratum. We also demonstrate that the miR expression profiles of FFPE samples are comparable to those of fresh-frozen samples, with a correlation of up to 0.87 (p Conclusion Our study thus demonstrates the utility, reproducibility, and optimization steps needed in miR expression studies using FFPE samples on a high-throughput quantitative PCR-based miR platform, opening up a realm of research possibilities for retrospective studies.

Selection of reference genes for quantitative real time RT-PCR during dimorphism in the zygomycete Mucor circinelloides.

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Valle-Maldonado, Marco I; Jácome-Galarza, Irvin E; Gutiérrez-Corona, Félix; Ramírez-Díaz, Martha I; Campos-García, Jesús; Meza-Carmen, Víctor

2015-03-01

Mucor circinelloides is a dimorphic fungal model for studying several biological processes including cell differentiation (yeast-mold transitions) as well as biodiesel and carotene production. The recent release of the first draft sequence of the M. circinelloides genome, combined with the availability of analytical methods to determine patterns of gene expression, such as quantitative Reverse transcription-Polymerase chain reaction (qRT-PCR), and the development of molecular genetic tools for the manipulation of the fungus, may help identify M. circinelloides gene products and analyze their relevance in different biological processes. However, no information is available on M. circinelloides genes of stable expression that could serve as internal references in qRT-PCR analyses. One approach to solve this problem consists in the use of housekeeping genes as internal references. However, validation of the usability of these reference genes is a fundamental step prior to initiating qRT-PCR assays. This work evaluates expression of several constitutive genes by qRT-PCR throughout the morphological differentiation stages of M. circinelloides; our results indicate that tfc-1 and ef-1 are the most stable genes for qRT-PCR assays during differentiation studies and they are proposed as reference genes to carry out gene expression studies in this fungus.

Identification of a set of endogenous reference genes for miRNA expression studies in Parkinson's disease blood samples.

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Serafin, Alice; Foco, Luisa; Blankenburg, Hagen; Picard, Anne; Zanigni, Stefano; Zanon, Alessandra; Pramstaller, Peter P; Hicks, Andrew A; Schwienbacher, Christine

2014-10-10

Research on microRNAs (miRNAs) is becoming an increasingly attractive field, as these small RNA molecules are involved in several physiological functions and diseases. To date, only few studies have assessed the expression of blood miRNAs related to Parkinson's disease (PD) using microarray and quantitative real-time PCR (qRT-PCR). Measuring miRNA expression involves normalization of qRT-PCR data using endogenous reference genes for calibration, but their choice remains a delicate problem with serious impact on the resulting expression levels. The aim of the present study was to evaluate the suitability of a set of commonly used small RNAs as normalizers and to identify which of these miRNAs might be considered reliable reference genes in qRT-PCR expression analyses on PD blood samples. Commonly used reference genes snoRNA RNU24, snRNA RNU6B, snoRNA Z30 and miR-103a-3p were selected from the literature. We then analyzed the effect of using these genes as reference, alone or in any possible combination, on the measured expression levels of the target genes miR-30b-5p and miR-29a-3p, which have been previously reported to be deregulated in PD blood samples. We identified RNU24 and Z30 as a reliable and stable pair of reference genes in PD blood samples.

Validation of commonly used reference genes for sleep-related gene expression studies

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Castro Rosa MRPS

2009-05-01

Full Text Available Abstract Background Sleep is a restorative process and is essential for maintenance of mental and physical health. In an attempt to understand the complexity of sleep, multidisciplinary strategies, including genetic approaches, have been applied to sleep research. Although quantitative real time PCR has been used in previous sleep-related gene expression studies, proper validation of reference genes is currently lacking. Thus, we examined the effect of total or paradoxical sleep deprivation (TSD or PSD on the expression stability of the following frequently used reference genes in brain and blood: beta-actin (b-actin, beta-2-microglobulin (B2M, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, and hypoxanthine guanine phosphoribosyl transferase (HPRT. Results Neither TSD nor PSD affected the expression stability of all tested genes in both tissues indicating that b-actin, B2M, GAPDH and HPRT are appropriate reference genes for the sleep-related gene expression studies. In order to further verify these results, the relative expression of brain derived neurotrophic factor (BDNF and glycerol-3-phosphate dehydrogenase1 (GPD1 was evaluated in brain and blood, respectively. The normalization with each of four reference genes produced similar pattern of expression in control and sleep deprived rats, but subtle differences in the magnitude of expression fold change were observed which might affect the statistical significance. Conclusion This study demonstrated that sleep deprivation does not alter the expression stability of commonly used reference genes in brain and blood. Nonetheless, the use of multiple reference genes in quantitative RT-PCR is required for the accurate results.

Selection of Reliable Reference Genes for Gene Expression Studies on Rhododendron molle G. Don.

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Xiao, Zheng; Sun, Xiaobo; Liu, Xiaoqing; Li, Chang; He, Lisi; Chen, Shangping; Su, Jiale

2016-01-01

The quantitative real-time polymerase chain reaction (qRT-PCR) approach has become a widely used method to analyze expression patterns of target genes. The selection of an optimal reference gene is a prerequisite for the accurate normalization of gene expression in qRT-PCR. The present study constitutes the first systematic evaluation of potential reference genes in Rhododendron molle G. Don. Eleven candidate reference genes in different tissues and flowers at different developmental stages of R. molle were assessed using the following three software packages: GeNorm, NormFinder, and BestKeeper. The results showed that EF1- α (elongation factor 1-alpha), 18S (18s ribosomal RNA), and RPL3 (ribosomal protein L3) were the most stable reference genes in developing rhododendron flowers and, thus, in all of the tested samples, while tublin ( TUB ) was the least stable. ACT5 (actin), RPL3 , 18S , and EF1- α were found to be the top four choices for different tissues, whereas TUB was not found to favor qRT-PCR normalization in these tissues. Three stable reference genes are recommended for the normalization of qRT-PCR data in R. molle . Furthermore, the expression profiles of RmPSY (phytoene synthase) and RmPDS (phytoene dehydrogenase) were assessed using EF1- α, 18S , ACT5 , RPL3 , and their combination as internals. Similar trends were found, but these trends varied when the least stable reference gene TUB was used. The results further prove that it is necessary to validate the stability of reference genes prior to their use for normalization under different experimental conditions. This study provides useful information for reliable qRT-PCR data normalization in gene studies of R. molle .

Selection of Reliable Reference Genes for Gene Expression Studies on Rhododendron molle G. Don

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Zheng Xiao

2016-10-01

Full Text Available The quantitative real-time polymerase chain reaction (qRT-PCR approach has become a widely used method to analyze expression patterns of target genes. The selection of an optimal reference gene is a prerequisite for the accurate normalization of gene expression in qRT-PCR. The present study constitutes the first systematic evaluation of potential reference genes in Rhododendron molle G. Don. Eleven candidate reference genes in different tissues and flowers at different developmental stages of R. molle were assessed using the following three software packages: GeNorm, NormFinder and BestKeeper. The results showed that EF1-α (elongation factor 1-alpha, 18S (18s ribosomal RNA and RPL3 (ribosomal protein L3 were the most stable reference genes in developing rhododendron flowers and, thus, in all of the tested samples, while tublin (TUB was the least stable. ACT5 (actin, RPL3, 18S and EF1-α were found to be the top four choices for different tissues, whereas TUB was not found to favor qRT-PCR normalization in these tissues. Three stable reference genes are recommended for the normalization of qRT-PCR data in R. molle. Furthermore, the expression profiles of RmPSY (phytoene synthase and RmPDS (phytoene dehydrogenase were assessed using EF1-α, 18S, ACT5, and RPL3 and their combination as internals. Similar trends were found, but these trends varied when the least stable reference gene TUB was used. The results further prove that it is necessary to validate the stability of reference genes prior to their use for normalization under different experimental conditions. This study provides useful information for reliable qRT-PCR data normalization in gene studies of R. molle.

Evaluation of PCR and multiplex PCR in relation to nested PCR for diagnosing Theileria equi

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Danielle C. Leal

2011-07-01

Full Text Available Conventional PCR (PCRTeq for diagnosing Theileria equi and multiplex PCR (M/PCRTeq-Bc for diagnosing T. equi and Babesia caballi were comparatively evaluated with nested PCR (N/PCR-Teq for diagnosing equine piroplasmosis. In DNA sensitivity determinations, in multiple dilutions of equine blood that had tested positive for T. equi, PCR-Teq and N/PCR-Teq detected hemoparasite DNA in the larger dilutions (1:128, but did not differ significantly from the M/PCRTeq-Bc (1:64. In analyses on equine serum tested by ELISA, there was high agreement between this serological test and PCR-Teq (k = 0.780 and moderate agreement with N/PCR-Teq (k = 0.562 and M/PCRTeq-Bc (k = 0.488. PCR-Teq found a higher frequency of T. equi both in extensively and intensively reared horses, but this was not significant in relation to N/PCR-Teq (P>0.05, and both PCRs indicated that there was an endemic situation regarding T. equi in the population of horses of this sample. PCR-Teq was only significantly different from M/PCR-Teq-Bc (P<0.05. PCR-Teq presented high sensitivity and specificity, comparable to N/PCR-Teq, but with the advantage of higher speed in obtaining results and lower costs and risks of laboratory contamination. This accredits PCR-Teq for epidemiological studies and for determinations on affected horses.

Validation of endogenous reference genes for qRT-PCR analysis of human visceral adipose samples.

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Mehta, Rohini; Birerdinc, Aybike; Hossain, Noreen; Afendy, Arian; Chandhoke, Vikas; Younossi, Zobair; Baranova, Ancha

2010-05-21

Given the epidemic proportions of obesity worldwide and the concurrent prevalence of metabolic syndrome, there is an urgent need for better understanding the underlying mechanisms of metabolic syndrome, in particular, the gene expression differences which may participate in obesity, insulin resistance and the associated series of chronic liver conditions. Real-time PCR (qRT-PCR) is the standard method for studying changes in relative gene expression in different tissues and experimental conditions. However, variations in amount of starting material, enzymatic efficiency and presence of inhibitors can lead to quantification errors. Hence the need for accurate data normalization is vital. Among several known strategies for data normalization, the use of reference genes as an internal control is the most common approach. Recent studies have shown that both obesity and presence of insulin resistance influence an expression of commonly used reference genes in omental fat. In this study we validated candidate reference genes suitable for qRT-PCR profiling experiments using visceral adipose samples from obese and lean individuals. Cross-validation of expression stability of eight selected reference genes using three popular algorithms, GeNorm, NormFinder and BestKeeper found ACTB and RPII as most stable reference genes. We recommend ACTB and RPII as stable reference genes most suitable for gene expression studies of human visceral adipose tissue. The use of these genes as a reference pair may further enhance the robustness of qRT-PCR in this model system.

Validation of endogenous reference genes for qRT-PCR analysis of human visceral adipose samples

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Afendy Arian

2010-05-01

Full Text Available Abstract Background Given the epidemic proportions of obesity worldwide and the concurrent prevalence of metabolic syndrome, there is an urgent need for better understanding the underlying mechanisms of metabolic syndrome, in particular, the gene expression differences which may participate in obesity, insulin resistance and the associated series of chronic liver conditions. Real-time PCR (qRT-PCR is the standard method for studying changes in relative gene expression in different tissues and experimental conditions. However, variations in amount of starting material, enzymatic efficiency and presence of inhibitors can lead to quantification errors. Hence the need for accurate data normalization is vital. Among several known strategies for data normalization, the use of reference genes as an internal control is the most common approach. Recent studies have shown that both obesity and presence of insulin resistance influence an expression of commonly used reference genes in omental fat. In this study we validated candidate reference genes suitable for qRT-PCR profiling experiments using visceral adipose samples from obese and lean individuals. Results Cross-validation of expression stability of eight selected reference genes using three popular algorithms, GeNorm, NormFinder and BestKeeper found ACTB and RPII as most stable reference genes. Conclusions We recommend ACTB and RPII as stable reference genes most suitable for gene expression studies of human visceral adipose tissue. The use of these genes as a reference pair may further enhance the robustness of qRT-PCR in this model system.

Selection and validation of a set of reliable reference genes for quantitative RT-PCR studies in the brain of the Cephalopod Mollusc Octopus vulgaris

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Biffali Elio

2009-07-01

Full Text Available Abstract Background Quantitative real-time polymerase chain reaction (RT-qPCR is valuable for studying the molecular events underlying physiological and behavioral phenomena. Normalization of real-time PCR data is critical for a reliable mRNA quantification. Here we identify reference genes to be utilized in RT-qPCR experiments to normalize and monitor the expression of target genes in the brain of the cephalopod mollusc Octopus vulgaris, an invertebrate. Such an approach is novel for this taxon and of advantage in future experiments given the complexity of the behavioral repertoire of this species when compared with its relatively simple neural organization. Results We chose 16S, and 18S rRNA, actB, EEF1A, tubA and ubi as candidate reference genes (housekeeping genes, HKG. The expression of 16S and 18S was highly variable and did not meet the requirements of candidate HKG. The expression of the other genes was almost stable and uniform among samples. We analyzed the expression of HKG into two different set of animals using tissues taken from the central nervous system (brain parts and mantle (here considered as control tissue by BestKeeper, geNorm and NormFinder. We found that HKG expressions differed considerably with respect to brain area and octopus samples in an HKG-specific manner. However, when the mantle is treated as control tissue and the entire central nervous system is considered, NormFinder revealed tubA and ubi as the most suitable HKG pair. These two genes were utilized to evaluate the relative expression of the genes FoxP, creb, dat and TH in O. vulgaris. Conclusion We analyzed the expression profiles of some genes here identified for O. vulgaris by applying RT-qPCR analysis for the first time in cephalopods. We validated candidate reference genes and found the expression of ubi and tubA to be the most appropriate to evaluate the expression of target genes in the brain of different octopuses. Our results also underline the

Identification and validation of reference genes for qRT-PCR studies of the obligate aphid pathogenic fungus Pandora neoaphidis during different developmental stages.

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Shutao Zhang

Full Text Available The selection of stable reference genes is a critical step for the accurate quantification of gene expression. To identify and validate the reference genes in Pandora neoaphidis-an obligate aphid pathogenic fungus-the expression of 13classical candidate reference genes were evaluated by quantitative real-time reverse transcriptase polymerase chain reaction(qPCR at four developmental stages (conidia, conidia with germ tubes, short hyphae and elongated hyphae. Four statistical algorithms, including geNorm, NormFinder, BestKeeper and Delta Ct method were used to rank putative reference genes according to their expression stability and indicate the best reference gene or combination of reference genes for accurate normalization. The analysis of comprehensive ranking revealed that ACT1and 18Swas the most stably expressed genes throughout the developmental stages. To further validate the suitability of the reference genes identified in this study, the expression of cell division control protein 25 (CDC25 and Chitinase 1(CHI1 genes were used to further confirm the validated candidate reference genes. Our study presented the first systematic study of reference gene(s selection for P. neoaphidis study and provided guidelines to obtain more accurate qPCR results for future developmental efforts.

Identification of Optimal Reference Genes for Normalization of qPCR Analysis during Pepper Fruit Development

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Yuan Cheng

2017-06-01

Full Text Available Due to its high sensitivity and reproducibility, quantitative real-time PCR (qPCR is practiced as a useful research tool for targeted gene expression analysis. For qPCR operations, the normalization with suitable reference genes (RGs is a crucial step that eventually determines the reliability of the obtained results. Although pepper is considered an ideal model plant for the study of non-climacteric fruit development, at present no specific RG have been developed or validated for the qPCR analyses of pepper fruit. Therefore, this study aimed to identify stably expressed genes for their potential use as RGs in pepper fruit studies. Initially, a total of 35 putative RGs were selected by mining the pepper transcriptome data sets derived from the PGP (Pepper Genome Platform and PGD (Pepper Genome Database. Their expression stabilities were further measured in a set of pepper (Capsicum annuum L. var. 007e fruit samples, which represented four different fruit developmental stages (IM: Immature; MG: Mature green; B: Break; MR: Mature red using the qPCR analysis. Then, based on the qPCR results, three different statistical algorithms, namely geNorm, Normfinder, and boxplot, were chosen to evaluate the expression stabilities of these putative RGs. It should be noted that nine genes were proven to be qualified as RGs during pepper fruit development, namely CaREV05 (CA00g79660; CaREV08 (CA06g02180; CaREV09 (CA06g05650; CaREV16 (Capana12g002666; CaREV21 (Capana10g001439; CaREV23 (Capana05g000680; CaREV26 (Capana01g002973; CaREV27 (Capana11g000123; CaREV31 (Capana04g002411; and CaREV33 (Capana08g001826. Further analysis based on geNorm suggested that the application of the two most stably expressed genes (CaREV05 and CaREV08 would provide optimal transcript normalization in the qPCR experiments. Therefore, a new and comprehensive strategy for the identification of optimal RGs was developed. This strategy allowed for the effective normalization of the qPCR

Expression patterns of WRKY genes in di-haploid Populus simonii × P. nigra in response to salinity stress revealed by quantitative real-time PCR and RNA sequencing.

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Wang, Shengji; Wang, Jiying; Yao, Wenjing; Zhou, Boru; Li, Renhua; Jiang, Tingbo

2014-10-01

Spatio-temporal expression patterns of 13 out of 119 poplar WRKY genes indicated dynamic and tissue-specific roles of WRKY family proteins in salinity stress tolerance. To understand the expression patterns of poplar WRKY genes under salinity stress, 51 of the 119 WRKY genes were selected from di-haploid Populus simonii × P. nigra by quantitative real-time PCR (qRT-PCR). We used qRT-PCR to profile the expression of the top 13 genes under salinity stress across seven time points, and employed RNA-Seq platforms to cross-validate it. Results demonstrated that all the 13 WRKY genes were expressed in root, stem, and leaf tissues, but their expression levels and overall patterns varied notably in these tissues. Regarding overall gene expression in roots, the 13 genes were significantly highly expressed at all six time points after the treatment, reaching the plateau of expression at hour 9. In leaves, the 13 genes were similarly up-regulated from 3 to 12 h in response to NaCl treatment. In stems, however, expression levels of the 13 genes did not show significant changes after the NaCl treatment. Regarding individual gene expression across the time points and the three tissues, the 13 genes can be classified into three clusters: the lowly expressed Cluster 1 containing PthWRKY28, 45 and 105; intermediately expressed Clusters 2 including PthWRKY56, 88 and 116; and highly expressed Cluster 3 consisting of PthWRKY41, 44, 51, 61, 62, 75 and 106. In general, genes in Cluster 2 and 3 displayed a dynamic pattern of "induced amplification-recovering", suggesting that these WRKY genes and corresponding pathways may play a critical role in mediating salt response and tolerance in a dynamic and tissue-specific manner.

Altered Gene Expression by Low-Dose Arsenic Exposure in Humans and Cultured Cardiomyocytes: Assessment by Real-Time PCR Arrays

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Judy Mumford

2011-06-01

Full Text Available Chronic arsenic exposure results in higher risk of skin, lung, and bladder cancer, as well as cardiovascular disease and diabetes. The purpose of this study was to investigate the effects on expression of selected genes in the blood lymphocytes from 159 people exposed chronically to arsenic in their drinking water using a novel RT-PCR TaqMan low-density array (TLDA. We found that expression of tumor necrosis factor-α (TNF-α, which activates both inflammation and NF-κB-dependent survival pathways, was strongly associated with water and urinary arsenic levels. Expression of KCNA5, which encodes a potassium ion channel protein, was positively associated with water and toe nail arsenic levels. Expression of 2 and 11 genes were positively associated with nail and urinary arsenic, respectively. Because arsenic exposure has been reported to be associated with long QT intervals and vascular disease in humans, we also used this TLDA for analysis of gene expression in human cardiomyocytes exposed to arsenic in vitro. Expression of the ion-channel genes CACNA1, KCNH2, KCNQ1 and KCNE1 were down-regulated by 1-mM arsenic. Alteration of some common pathways, including those involved in oxidative stress, inflammatory signaling, and ion-channel function, may underlay the seemingly disparate array of arsenic-associated diseases, such as cancer, cardiovascular disease, and diabetes.

Selection and validation of reference genes for miRNA expression studies during porcine pregnancy.

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Jocelyn M Wessels

Full Text Available MicroRNAs comprise a family of small non-coding RNAs that modulate several developmental and physiological processes including pregnancy. Their ubiquitous presence is confirmed in mammals, worms, flies and plants. Although rapid advances have been made in microRNA research, information on stable reference genes for validation of microRNA expression is still lacking. Real time PCR is a widely used tool to quantify gene transcripts. An appropriate reference gene must be chosen to minimize experimental error in this system. A small difference in miRNA levels between experimental samples can be biologically meaningful as these entities can affect multiple targets in a pathway. This study examined the suitability of six commercially available reference genes (RNU1A, RNU5A, RNU6B, SNORD25, SCARNA17, and SNORA73A in maternal-fetal tissues from healthy and spontaneously arresting/dying conceptuses from sows were separately analyzed at gestation day 20. Comparisons were also made with non-pregnant endometrial tissues from sows. Spontaneous fetal loss is a prime concern to the commercial pork industry. Our laboratory has previously identified deficits in vasculature development at maternal-fetal interface as one of the major participating causes of fetal loss. Using this well-established model, we have extended our studies to identify suitable microRNA reference genes. A methodical approach to assessing suitability was adopted using standard curve and melting curve analysis, PCR product sequencing, real time PCR expression in a panel of gestational tissues, and geNorm and NormFinder analysis. Our quantitative real time PCR analysis confirmed expression of all 6 reference genes in maternal and fetal tissues. All genes were uniformly expressed in tissues from healthy and spontaneously arresting conceptus attachment sites. Comparisons between tissue types (maternal/fetal/non-pregnant revealed significant differences for RNU5A, RNU6B, SCARNA17, and SNORA73A

Identification of stable reference genes for quantitative PCR in cells derived from chicken lymphoid organs.

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Borowska, D; Rothwell, L; Bailey, R A; Watson, K; Kaiser, P

2016-02-01

Quantitative polymerase chain reaction (qPCR) is a powerful technique for quantification of gene expression, especially genes involved in immune responses. Although qPCR is a very efficient and sensitive tool, variations in the enzymatic efficiency, quality of RNA and the presence of inhibitors can lead to errors. Therefore, qPCR needs to be normalised to obtain reliable results and allow comparison. The most common approach is to use reference genes as internal controls in qPCR analyses. In this study, expression of seven genes, including β-actin (ACTB), β-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-glucuronidase (GUSB), TATA box binding protein (TBP), α-tubulin (TUBAT) and 28S ribosomal RNA (r28S), was determined in cells isolated from chicken lymphoid tissues and stimulated with three different mitogens. The stability of the genes was measured using geNorm, NormFinder and BestKeeper software. The results from both geNorm and NormFinder were that the three most stably expressed genes in this panel were TBP, GAPDH and r28S. BestKeeper did not generate clear answers because of the highly heterogeneous sample set. Based on these data we will include TBP in future qPCR normalisation. The study shows the importance of appropriate reference gene normalisation in other tissues before qPCR analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

[Detecting HB-1 Expression Level in Bone Marrow of Acute Leukemia Patients by Real-Time Fluorescence Quantitative RT-PCR].

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Wang, Qing-Yun; Li, Yuan; Ji, Li; Liang, Ze-Yin; Liu, Wei; Ren, Han-Yun; Qiu, Zhi-Xiang

2018-02-01

To investigate the expression level of HB-1 gene in patients with acute lymphoblastic leukemia (ALL) and the significance of HB-1 gene in monitoring of minimal residual disease (MRD). The method of real-time fluorescence quantitative RT-PCR (Taqman probe) was established to detect the expression levels of HB-1 gene; then the sensitivity, specificity and repeatability of this assay were evaluated and verified. The HB-1 gene expression levels in bone marrow of 183 cases of ALL, 70 cases of acute myeloid leukemias (AML), 52 cases of non-malignant hematologic diseases and 24 healthy hematopoietic stem cell donors were detected. The correlation of HB-1 level with diagnosis and relapse was analyzed by detecting bone marrow samples of 33 B-ALL. The sensitivity of this assay reached the 10 -4 level. The coefficient of variation for inter-batch and inter-tube of HB-1 were 6.79% and 4.80%, respectively. It was found that HB-1 gene specifically expressed in acute B lymphoblastic leukemia. The median expression levels of HB-1 gene in newly diagnosed and relapsed B-ALL patients were statistically significantly higher than those in ALL in complete remission(CR), newly diagnosed T-ALL, newly diagnosed AML, non-malignant hematologic diseases, and healthy hematopoietic stem cell donors(33.0% vs 0.68%, 0.07%, 0.02%, 0.58% and 0, respectively) (P0.05). The expression level of HB-1 gene declined sharply when B-ALL patients reached complete remission (0-7.99%, with median level 0.68%), but increased when relapsed (7.69%, 8.08% and 484.0% in 3 relapsed samples), which was in accordance with results of flow cytometry. HB-1 gene specifically expressed in acute B lymphoblastic leukemia cells. The established real-time fluorescence quantitative RT-PCR assay shows good sensitivity, specificity and repeatability, thus, can be used as a biological marker in the clinical detection, monitoring MRD and predicting of early relapse for B-ALL patients.

Selection of reference genes for gene expression studies in heart failure for left and right ventricles.

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Li, Mengmeng; Rao, Man; Chen, Kai; Zhou, Jianye; Song, Jiangping

2017-07-15

Real-time quantitative reverse transcriptase-PCR (qRT-PCR) is a feasible tool for determining gene expression profiles, but the accuracy and reliability of the results depends on the stable expression of selected housekeeping genes in different samples. By far, researches on stable housekeeping genes in human heart failure samples are rare. Moreover the effect of heart failure on the expression of housekeeping genes in right and left ventricles is yet to be studied. Therefore we aim to provide stable housekeeping genes for both ventricles in heart failure and normal heart samples. In this study, we selected seven commonly used housekeeping genes as candidates. By using the qRT-PCR, the expression levels of ACTB, RAB7A, GAPDH, REEP5, RPL5, PSMB4 and VCP in eight heart failure and four normal heart samples were assessed. The stability of candidate housekeeping genes was evaluated by geNorm and Normfinder softwares. GAPDH showed the least variation in all heart samples. Results also indicated the difference of gene expression existed in heart failure left and right ventricles. GAPDH had the highest expression stability in both heart failure and normal heart samples. We also propose using different sets of housekeeping genes for left and right ventricles respectively. The combination of RPL5, GAPDH and PSMB4 is suitable for the right ventricle and the combination of GAPDH, REEP5 and RAB7A is suitable for the left ventricle. Copyright © 2017 Elsevier B.V. All rights reserved.

Normalization of Reverse Transcription Quantitative PCR Data During Ageing in Distinct Cerebral Structures.

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Bruckert, G; Vivien, D; Docagne, F; Roussel, B D

2016-04-01

Reverse transcription quantitative-polymerase chain reaction (RT-qPCR) has become a routine method in many laboratories. Normalization of data from experimental conditions is critical for data processing and is usually achieved by the use of a single reference gene. Nevertheless, as pointed by the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, several reference genes should be used for reliable normalization. Ageing is a physiological process that results in a decline of many expressed genes. Reliable normalization of RT-qPCR data becomes crucial when studying ageing. Here, we propose a RT-qPCR study from four mouse brain regions (cortex, hippocampus, striatum and cerebellum) at different ages (from 8 weeks to 22 months) in which we studied the expression of nine commonly used reference genes. With the use of two different algorithms, we found that all brain structures need at least two genes for a good normalization step. We propose specific pairs of gene for efficient data normalization in the four brain regions studied. These results underline the importance of reliable reference genes for specific brain regions in ageing.

Evaluation of reference genes for RT-qPCR study in abalone Haliotis discus hannai during heavy metal overload stress

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Sang Yoon Lee

2016-06-01

Full Text Available Abstract Background The evaluation of suitable reference genes as normalization controls is a prerequisite requirement for launching quantitative reverse transcription-PCR (RT-qPCR-based expression study. In order to select the stable reference genes in abalone Haliotis discus hannai tissues (gill and hepatopancreas under heavy metal exposure conditions (Cu, Zn, and Cd, 12 potential candidate housekeeping genes were subjected to expression stability based on the comprehensive ranking while integrating four different statistical algorithms (geNorm, NormFinder, BestKeeper, and ΔCT method. Results Expression stability in the gill subset was determined as RPL7 > RPL8 > ACTB > RPL3 > PPIB > RPL7A > EF1A > RPL4 > GAPDH > RPL5 > UBE2 > B-TU. On the other hand, the ranking in the subset for hepatopancreas was RPL7 > RPL3 > RPL8 > ACTB > RPL4 > EF1A > RPL5 > RPL7A > B-TU > UBE2 > PPIB > GAPDH. The pairwise variation assessed by the geNorm program indicates that two reference genes could be sufficient for accurate normalization in both gill and hepatopancreas subsets. Overall, both gill and hepatopancreas subsets recommended ribosomal protein genes (particularly RPL7 as stable references, whereas traditional housekeepers such as β-tubulin (B-TU and glyceraldehyde-3-phosphate dehydrogenase (GAPDH genes were ranked as unstable genes. The validation of reference gene selection was confirmed with the quantitative assay of MT transcripts. Conclusions The present analysis showed the importance of validating reference genes with multiple algorithmic approaches to select genes that are truly stable. Our results indicate that expression stability of a given reference gene could not always have consensus across tissue types. The data from this study could be a good guide for the future design of RT-qPCR studies with respect to metal regulation/detoxification and other related

Modeling qRT-PCR dynamics with application to cancer biomarker quantification.

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Chervoneva, Inna; Freydin, Boris; Hyslop, Terry; Waldman, Scott A

2017-01-01

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is widely used for molecular diagnostics and evaluating prognosis in cancer. The utility of mRNA expression biomarkers relies heavily on the accuracy and precision of quantification, which is still challenging for low abundance transcripts. The critical step for quantification is accurate estimation of efficiency needed for computing a relative qRT-PCR expression. We propose a new approach to estimating qRT-PCR efficiency based on modeling dynamics of polymerase chain reaction amplification. In contrast, only models for fluorescence intensity as a function of polymerase chain reaction cycle have been used so far for quantification. The dynamics of qRT-PCR efficiency is modeled using an ordinary differential equation model, and the fitted ordinary differential equation model is used to obtain effective polymerase chain reaction efficiency estimates needed for efficiency-adjusted quantification. The proposed new qRT-PCR efficiency estimates were used to quantify GUCY2C (Guanylate Cyclase 2C) mRNA expression in the blood of colorectal cancer patients. Time to recurrence and GUCY2C expression ratios were analyzed in a joint model for survival and longitudinal outcomes. The joint model with GUCY2C quantified using the proposed polymerase chain reaction efficiency estimates provided clinically meaningful results for association between time to recurrence and longitudinal trends in GUCY2C expression.

Downregulated miR-23b-3p expression acts as a predictor of hepatocellular carcinoma progression: A study based on public data and RT-qPCR verification

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Xiang, Xue-Lian; Yang, Xia; Liang, Hai-Wei; Qiu, Xiao-Hui; Yang, Li-Hua; Peng, Zhi-Gang; Chen, Gang

2018-01-01

Mounting evidence has shown that miR-23b-3p, which is associated with cell proliferation, invasion, and apoptosis, acts as a biomarker for diagnosis and outcomes in numerous cancers. However, the clinicopathological implication of miR-23b-3p in hepatocellular carcinoma (HCC) remains unclear. Our study evaluated the role of miR-23b-3p in HCC and investigated its potential application as a marker for preliminary diagnosis and therapy in HCC. High-throughput data from the NCBI Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) were collected and analyzed. One hundred and one tissue sections of HCC were paired with adjacent non-cancerous HCC as further supplements. miR-23b-3p expression was detected using quantitative real-time PCR. Additionally, the relationship between miR-23b-3p expression and HCC progression and Time-to-recurrence (months) was explored. Ten algorithms were applied to predict the prospective target genes of miR-23b-3p. Next, we conducted bioinformatics analysis for further study. miR-23b-3p expression was pronouncedly decreased in HCC tissues in contrast with their paired adjacent non-cancerous HCC (PAKT1, EGFR, CTNNB1, BCL2, SMAD3, PTEN and KDM6A, were located in the key nodes with high degree (>35). In conclusion, this study provides impressive illumination of the potential role of miR-23b-3p in HCC tumorigenesis and progression. Furthermore, miR-23b-3p may act as a predictor of HCC and could be a new treatment target. PMID:29484429

Validation of Reference Genes for Quantitative Expression Analysis by Real-Time RT-PCR in Four Lepidopteran Insects

OpenAIRE

Teng, Xiaolu; Zhang, Zan; He, Guiling; Yang, Liwen; Li, Fei

2012-01-01

Quantitative real-time polymerase chain reaction (qPCR) is an efficient and widely used technique to monitor gene expression. Housekeeping genes (HKGs) are often empirically selected as the reference genes for data normalization. However, the suitability of HKGs used as the reference genes has been seldom validated. Here, six HKGs were chosen (actin A3, actin A1, GAPDH, G3PDH, E2F, rp49) in four lepidopteran insects Bombyx mori L. (Lepidoptera: Bombycidae), Plutella xylostella L. (Plutellidae...

Real-time PCR expression profiling of genes encoding potential virulence factors in Candida albicans biofilms: identification of model-dependent and -independent gene expression

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Řičicová Markéta

2010-04-01

Full Text Available Abstract Background Candida albicans infections are often associated with biofilm formation. Previous work demonstrated that the expression of HWP1 (hyphal wall protein and of genes belonging to the ALS (agglutinin-like sequence, SAP (secreted aspartyl protease, PLB (phospholipase B and LIP (lipase gene families is associated with biofilm growth on mucosal surfaces. We investigated using real-time PCR whether genes encoding potential virulence factors are also highly expressed in biofilms associated with abiotic surfaces. For this, C. albicans biofilms were grown on silicone in microtiter plates (MTP or in the Centres for Disease Control (CDC reactor, on polyurethane in an in vivo subcutaneous catheter rat (SCR model, and on mucosal surfaces in the reconstituted human epithelium (RHE model. Results HWP1 and genes belonging to the ALS, SAP, PLB and LIP gene families were constitutively expressed in C. albicans biofilms. ALS1-5 were upregulated in all model systems, while ALS9 was mostly downregulated. ALS6 and HWP1 were overexpressed in all models except in the RHE and MTP, respectively. The expression levels of SAP1 were more pronounced in both in vitro models, while those of SAP2, SAP4 and SAP6 were higher in the in vivo model. Furthermore, SAP5 was highly upregulated in the in vivo and RHE models. For SAP9 and SAP10 similar gene expression levels were observed in all model systems. PLB genes were not considerably upregulated in biofilms, while LIP1-3, LIP5-7 and LIP9-10 were highly overexpressed in both in vitro models. Furthermore, an elevated lipase activity was detected in supernatans of biofilms grown in the MTP and RHE model. Conclusions Our findings show that HWP1 and most of the genes belonging to the ALS, SAP and LIP gene families are upregulated in C. albicans biofilms. Comparison of the fold expression between the various model systems revealed similar expression levels for some genes, while for others model-dependent expression

With Reference to Reference Genes: A Systematic Review of Endogenous Controls in Gene Expression Studies.

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Chapman, Joanne R; Waldenström, Jonas

2015-01-01

The choice of reference genes that are stably expressed amongst treatment groups is a crucial step in real-time quantitative PCR gene expression studies. Recent guidelines have specified that a minimum of two validated reference genes should be used for normalisation. However, a quantitative review of the literature showed that the average number of reference genes used across all studies was 1.2. Thus, the vast majority of studies continue to use a single gene, with β-actin (ACTB) and/or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) being commonly selected in studies of vertebrate gene expression. Few studies (15%) tested a panel of potential reference genes for stability of expression before using them to normalise data. Amongst studies specifically testing reference gene stability, few found ACTB or GAPDH to be optimal, whereby these genes were significantly less likely to be chosen when larger panels of potential reference genes were screened. Fewer reference genes were tested for stability in non-model organisms, presumably owing to a dearth of available primers in less well characterised species. Furthermore, the experimental conditions under which real-time quantitative PCR analyses were conducted had a large influence on the choice of reference genes, whereby different studies of rat brain tissue showed different reference genes to be the most stable. These results highlight the importance of validating the choice of normalising reference genes before conducting gene expression studies.

Selection of reliable reference genes for quantitative real-time PCR in human T cells and neutrophils

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Ledderose Carola

2011-10-01

Full Text Available Abstract Background The choice of reliable reference genes is a prerequisite for valid results when analyzing gene expression with real-time quantitative PCR (qPCR. This method is frequently applied to study gene expression patterns in immune cells, yet a thorough validation of potential reference genes is still lacking for most leukocyte subtypes and most models of their in vitro stimulation. In the current study, we evaluated the expression stability of common reference genes in two widely used cell culture models-anti-CD3/CD28 activated T cells and lipopolysaccharide stimulated neutrophils-as well as in unselected untreated leukocytes. Results The mRNA expression of 17 (T cells, 7 (neutrophils or 8 (unselected leukocytes potential reference genes was quantified by reverse transcription qPCR, and a ranking of the preselected candidate genes according to their expression stability was calculated using the programs NormFinder, geNorm and BestKeeper. IPO8, RPL13A, TBP and SDHA were identified as suitable reference genes in T cells. TBP, ACTB and SDHA were stably expressed in neutrophils. TBP and SDHA were also the most stable genes in untreated total blood leukocytes. The critical impact of reference gene selection on the estimated target gene expression is demonstrated for IL-2 and FIH expression in T cells. Conclusions The study provides a shortlist of suitable reference genes for normalization of gene expression data in unstimulated and stimulated T cells, unstimulated and stimulated neutrophils and in unselected leukocytes.

Detection of ROS1 Gene Rearrangement in Lung Adenocarcinoma: Comparison of IHC, FISH and Real-Time RT-PCR

OpenAIRE

Shan, Ling; Lian, Fang; Guo, Lei; Qiu, Tian; Ling, Yun; Ying, Jianming; Lin, Dongmei

2015-01-01

Aims To compare fluorescence in situ hybridization (FISH), immunohistochemistry (IHC) and quantitative real-time reverse transcription-PCR (qRT-PCR) assays for detection of ROS1 fusion in a large number of ROS1-positive lung adenocatcinoma (ADC) patients. Methods Using IHC analysis, sixty lung ADCs including 16 cases with ROS1 protein expression and 44 cases without ROS1 expression were selected for this study. The ROS1 fusion status was examined by FISH and qRT-PCR assay. Results Among 60 ca...

Evaluation of reference genes for quantitative real-time PCR in oil palm elite planting materials propagated by tissue culture.

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Pek-Lan Chan

Full Text Available BACKGROUND: The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR. With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels. RESULTS: In this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569 outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN. PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization. CONCLUSIONS: Systematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection

Evaluation of Reference Genes for Quantitative Real-Time PCR in Oil Palm Elite Planting Materials Propagated by Tissue Culture

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Chan, Pek-Lan; Rose, Ray J.; Abdul Murad, Abdul Munir; Zainal, Zamri; Leslie Low, Eng-Ti; Ooi, Leslie Cheng-Li; Ooi, Siew-Eng; Yahya, Suzaini; Singh, Rajinder

2014-01-01

Background The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR). With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels. Results In this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper) confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569) outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN). PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization. Conclusions Systematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection of appropriate

RNA-seq reveals more consistent reference genes for gene expression studies in human non-melanoma skin cancers

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Van L.T. Hoang

2017-08-01

Full Text Available Identification of appropriate reference genes (RGs is critical to accurate data interpretation in quantitative real-time PCR (qPCR experiments. In this study, we have utilised next generation RNA sequencing (RNA-seq to analyse the transcriptome of a panel of non-melanoma skin cancer lesions, identifying genes that are consistently expressed across all samples. Genes encoding ribosomal proteins were amongst the most stable in this dataset. Validation of this RNA-seq data was examined using qPCR to confirm the suitability of a set of highly stable genes for use as qPCR RGs. These genes will provide a valuable resource for the normalisation of qPCR data for the analysis of non-melanoma skin cancer.

Reference gene selection for molecular studies of dormancy in wild oat (Avena fatua L. caryopses by RT-qPCR method.

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Izabela Ruduś

Full Text Available Molecular studies of primary and secondary dormancy in Avena fatua L., a serious weed of cereal and other crops, are intended to reveal the species-specific details of underlying molecular mechanisms which in turn may be useable in weed management. Among others, quantitative real-time PCR (RT-qPCR data of comparative gene expression analysis may give some insight into the involvement of particular wild oat genes in dormancy release, maintenance or induction by unfavorable conditions. To assure obtaining biologically significant results using this method, the expression stability of selected candidate reference genes in different data subsets was evaluated using four statistical algorithms i.e. geNorm, NormFinder, Best Keeper and ΔCt method. Although some discrepancies in their ranking outputs were noticed, evidently two ubiquitin-conjugating enzyme homologs, AfUBC1 and AfUBC2, as well as one homolog of glyceraldehyde 3-phosphate dehydrogenase AfGAPDH1 and TATA-binding protein AfTBP2 appeared as more stably expressed than AfEF1a (translation elongation factor 1α, AfGAPDH2 or the least stable α-tubulin homolog AfTUA1 in caryopses and seedlings of A. fatua. Gene expression analysis of a dormancy-related wild oat transcription factor VIVIPAROUS1 (AfVP1 allowed for a validation of candidate reference genes performance. Based on the obtained results it can be recommended that the normalization factor calculated as a geometric mean of Cq values of AfUBC1, AfUBC2 and AfGAPDH1 would be optimal for RT-qPCR results normalization in the experiments comprising A. fatua caryopses of different dormancy status.

Reference gene selection for molecular studies of dormancy in wild oat (Avena fatua L.) caryopses by RT-qPCR method.

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Ruduś, Izabela; Kępczyński, Jan

2018-01-01

Molecular studies of primary and secondary dormancy in Avena fatua L., a serious weed of cereal and other crops, are intended to reveal the species-specific details of underlying molecular mechanisms which in turn may be useable in weed management. Among others, quantitative real-time PCR (RT-qPCR) data of comparative gene expression analysis may give some insight into the involvement of particular wild oat genes in dormancy release, maintenance or induction by unfavorable conditions. To assure obtaining biologically significant results using this method, the expression stability of selected candidate reference genes in different data subsets was evaluated using four statistical algorithms i.e. geNorm, NormFinder, Best Keeper and ΔCt method. Although some discrepancies in their ranking outputs were noticed, evidently two ubiquitin-conjugating enzyme homologs, AfUBC1 and AfUBC2, as well as one homolog of glyceraldehyde 3-phosphate dehydrogenase AfGAPDH1 and TATA-binding protein AfTBP2 appeared as more stably expressed than AfEF1a (translation elongation factor 1α), AfGAPDH2 or the least stable α-tubulin homolog AfTUA1 in caryopses and seedlings of A. fatua. Gene expression analysis of a dormancy-related wild oat transcription factor VIVIPAROUS1 (AfVP1) allowed for a validation of candidate reference genes performance. Based on the obtained results it can be recommended that the normalization factor calculated as a geometric mean of Cq values of AfUBC1, AfUBC2 and AfGAPDH1 would be optimal for RT-qPCR results normalization in the experiments comprising A. fatua caryopses of different dormancy status.

Selection of Reference Genes for Expression Studies in Diaphorina citri (Hemiptera: Liviidae).

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Bassan, Meire Menezes; Angelotti-Mendonc A, Je Ssika; Alves, Gustavo Rodrigues; Yamamoto, Pedro Takao; Moura O Filho, Francisco de Assis Alves

2017-12-05

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is considered the main vector of the bacteria associated with huanglongbing, a very serious disease that has threatened the world citrus industry. The absence of efficient control management protocols, including a lack of resistant cultivars, has led to the development of different approaches to study this pathosystem. The production of resistant genotypes relies on D. citri gene expression analyses by RT-qPCR to assess control of the vector population. High-quality, reliable RT-qPCR analyses depend upon proper reference gene selection and validation. However, adequate D. citri reference genes have not yet been identified. In the present study, we evaluated the genes EF 1-α, ACT, GAPDH, RPL7, RPL17, and TUB as candidate reference genes for this insect. Gene expression stability was evaluated using the mathematical algorithms deltaCt, NormFinder, BestKeeper, and geNorm, at five insect developmental stages, grown on two different plant hosts [Citrus sinensis (L.) Osbeck (Sapindales: Rutaceae) and Murraya paniculata (L.) Jack (Sapindales: Rutaceae)]. The final gene ranking was calculated using RefFinder software, and the V-ATPase-A gene was selected for validation. According to our results, two reference genes are recommended when different plant hosts and developmental stages are considered. Considering gene expression studies in D. citri grown on M. paniculata, regardless of the insect developmental stage, GAPDH and RPL7 have the best fit as reference genes in RT-qPCR analyses, whereas GAPDH and EF 1-α are recommended as reference genes in insect studies using C. sinensis. © The Author(s) 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Real-time quantitative PCR of microdissected paraffin-embedded breast carcinoma

DEFF Research Database (Denmark)

Gjerdrum, Lise Mette; Sorensen, Boe Sandahl; Kjeldsen, Eigil

2004-01-01

We studied the feasibility of using real-time quantitative PCR to determine HER-2 DNA amplification and mRNA expression in microdissected formalin-fixed, paraffin-embedded breast tumors and compared this with standard immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) methods...... tumors as being amplified. Interestingly, all these scored 2+ with the HercepTest, but were negative using FISH. We believe that real-time quantitative PCR analysis of HER-2 DNA amplification following microdissection represents a useful supplementary or perhaps even an alternative technique...

Real-time quantitative PCR of microdissected paraffin-embedded breast carcinoma

DEFF Research Database (Denmark)

Gjerdrum, Lise Mette; Sorensen, Boe Sandahl; Kjeldsen, Eigil

2004-01-01

We studied the feasibility of using real-time quantitative PCR to determine HER-2 DNA amplification and mRNA expression in microdissected formalin-fixed, paraffin-embedded breast tumors and compared this with standard immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) methods...

Identification of suitable reference genes for gene expression studies of shoulder instability.

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Mariana Ferreira Leal

Full Text Available Shoulder instability is a common shoulder injury, and patients present with plastic deformation of the glenohumeral capsule. Gene expression analysis may be a useful tool for increasing the general understanding of capsule deformation, and reverse-transcription quantitative polymerase chain reaction (RT-qPCR has become an effective method for such studies. Although RT-qPCR is highly sensitive and specific, it requires the use of suitable reference genes for data normalization to guarantee meaningful and reproducible results. In the present study, we evaluated the suitability of a set of reference genes using samples from the glenohumeral capsules of individuals with and without shoulder instability. We analyzed the expression of six commonly used reference genes (ACTB, B2M, GAPDH, HPRT1, TBP and TFRC in the antero-inferior, antero-superior and posterior portions of the glenohumeral capsules of cases and controls. The stability of the candidate reference gene expression was determined using four software packages: NormFinder, geNorm, BestKeeper and DataAssist. Overall, HPRT1 was the best single reference gene, and HPRT1 and B2M composed the best pair of reference genes from different analysis groups, including simultaneous analysis of all tissue samples. GenEx software was used to identify the optimal number of reference genes to be used for normalization and demonstrated that the accumulated standard deviation resulting from the use of 2 reference genes was similar to that resulting from the use of 3 or more reference genes. To identify the optimal combination of reference genes, we evaluated the expression of COL1A1. Although the use of different reference gene combinations yielded variable normalized quantities, the relative quantities within sample groups were similar and confirmed that no obvious differences were observed when using 2, 3 or 4 reference genes. Consequently, the use of 2 stable reference genes for normalization, especially

Study On Application Of Molecular Techniques (RAPD-PCR And RAMP-PCR) To Detect Mutation In Rice Breeding

International Nuclear Information System (INIS)

Hoang Thi My Linh; Phan, D. T. Son; Nguyen Thi Vang; Nguyen, T. T. Hien; Le XuanTham

2007-01-01

The project was carried out in 2007 with the purpose of consideration for using the two simple and inexpensive molecular techniques to estimate changes in DNA of rice mutant after gamma irradiation. Three rice cultivars: Basmati370, Tam Thom (TT1), IR64 and three gamma irradiated mutants BDS, TDS and VND 95-20 respectively, were used. Suitable DNA extraction procedure was obtained. PCR optimization was conducted on three important factors including: amount of MgCl 2 , DNA concentration and annealing temperature. 2.5 mM of MgCl 2 for RAPD-PCR and 3.75 mM for RAMP-PCR were found the best. 40 ng DNA provided a good amplification for RAMP-PCR; this figure was 50 ng for RAPD-PCR. Annealing temperatures were determined at 36 o C for RAPD primer and at 55±3 o C for Microsatellite primer. Final results showed that, both RAPD-PCR and RAMP-PCR could detect changes in DNA of rice mutants after gamma irradiation compared to their parents. Percentage of DNA changes determined by RAPD-PCR and RAMP-PCR on Basmati370 and its mutant BDS were 11.49% and 21.2% respectively; These on TT1 and TDS were 8.98% and 15.4%; and on IR64 and VND 95-20 were 3.45% and 4.95%. (author)

Evaluation of Reference Genes for Reverse Transcription Quantitative PCR Studies of Physiological Responses in the Ghost Moth, Thitarodes armoricanus (Lepidoptera, Hepialidae.

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Guiqing Liu

Full Text Available Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR is the sensitive method to quantify the expression levels of target genes on the basis of endogenous control. An appropriate reference gene set for normalization is essential for reliable results. The ghost moth, Thitarodes armoricanus, a host species of a medicinal fungus, Ophiocordyceps sinensis, is an economically important member of the Lepidoptera. Recent studies have focused on the mechanism of adaptation of this species to its high-altitude environment and host immune response to O. sinensis infection and RT-qPCR is commonly used in these studies to decipher the genetic basis of physiological functions. However, a thorough assessment of candidate reference genes in the genus Thitarodes is lacking. Here, the expression levels of eight candidate reference genes (ACT, EF, EIF4A, GAPDH, G6PDH, RPL13A, TUB and 18S in T. armoricanus at different developmental stages and in different body parts of the seventh instar larvae were analyzed, along with larvae kept under low temperatures, larvae exposed to two fungal infections and larvae fed different diets. Three established software programs-Bestkeeper, geNorm and NormFinder-were employed to calculate variation among the treatments. The results revealed that the best-suited reference genes differed across the treatments, with EF, EIF4A and GAPDH found to be the best suited for the different developmental stages and larvae body parts; EF, EIF4A and RPL13A found to be the best suited for low-temperature challenge; and EF, EIF4A and TUB found to be the best suited for the fungal infections and dietary treatments. This study thus further contributes to the establishment of an accurate method for normalizing RT-qPCR results for T. armoricanus and serves as a reference for gene expression studies of related insect species.

Improved soluble expression and characterization of the Hc domain of Clostridium botulinum neurotoxin serotype A in Escherichia coli by using a PCR-synthesized gene and a Trx co-expression strain.

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Chen, Rongchang; Shi, Jing; Cai, Kun; Tu, Wei; Hou, Xiaojun; Liu, Hao; Xiao, Le; Wang, Qin; Tang, Yunming; Wang, Hui

2010-05-01

Botulinum neurotoxin serotype A (BoNT/A) is an extremely potent bacterial protein toxin. The Hc fragment of BoNT/A (AHc) was shown to be non-toxic, antigenic, and capable of eliciting a protective immunity in animals challenged with homologous BoNT. In this study, we synthesized AHc gene by using T4 DNA ligase and PCR. The AHc was expressed at a high level in Escherichia coli successfully. Because of using the Trx co-expression strain, the expressed AHc is in a soluble and active form. The yield of the purified AHc was about 70mg/L, and its purity was up to 90% through one-step affinity chromatography. The AHc was positively identified by the antibodies raised against BoNT/A using immunological-dot-blot and Western blot assays. AHc was shown to bind with gangliosides and elicit immunity against BoNT/A, indicating that the expressed and purified AHc protein retains a functionally active conformation. Furthermore, the purified AHc has a strong immunogenicity and can be used as a potential subunit candidate vaccine for botulinum toxin serotype A. Copyright (c) 2009 Elsevier Inc. All rights reserved.

Droplet digital PCR technology promises new applications and research areas.

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Manoj, P

2016-01-01

Digital Polymerase Chain Reaction (dPCR) is used to quantify nucleic acids and its applications are in the detection and precise quantification of low-level pathogens, rare genetic sequences, quantification of copy number variants, rare mutations and in relative gene expressions. Here the PCR is performed in large number of reaction chambers or partitions and the reaction is carried out in each partition individually. This separation allows a more reliable collection and sensitive measurement of nucleic acid. Results are calculated by counting amplified target sequence (positive droplets) and the number of partitions in which there is no amplification (negative droplets). The mean number of target sequences was calculated by Poisson Algorithm. Poisson correction compensates the presence of more than one copy of target gene in any droplets. The method provides information with accuracy and precision which is highly reproducible and less susceptible to inhibitors than qPCR. It has been demonstrated in studying variations in gene sequences, such as copy number variants and point mutations, distinguishing differences between expression of nearly identical alleles, assessment of clinically relevant genetic variations and it is routinely used for clonal amplification of samples for NGS methods. dPCR enables more reliable predictors of tumor status and patient prognosis by absolute quantitation using reference normalizations. Rare mitochondrial DNA deletions associated with a range of diseases and disorders as well as aging can be accurately detected with droplet digital PCR.

Expressão dos genes nodC, nodW e nopP em Bradyrhizobium japonicum estirpe CPAC 15 avaliada por RT-qPCR Expression of nodC, nodW and nopP genes in Bradyrhizobium japonicum CPAC 15 strain evaluated by RT-qPCR

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Simone Bortolan

2009-11-01

Full Text Available O objetivo deste trabalho foi avaliar a expressão, por RT-qPCR, dos genes de nodulação nodC e nodW e do gene nopP da estirpe CPAC 15, que provavelmente atuam na infecção das raízes da soja. Foram realizados dois experimentos. No primeiro, a expressão dos genes foi avaliada nas células após a incubação com genisteína por 15 min, 1, 4 e 8 horas. Os resultados revelaram que os três genes apresentaram maior expressão imediatamente após o contato com o indutor (15 min. No segundo experimento, a bactéria foi cultivada na presença de indutores (genisteína ou exsudatos de sementes de soja por 48 horas. A expressão dos três genes foi maior na presença de genisteína, com valores de expressão para nodC, nodW e nopP superiores ao controle. Os resultados obtidos confirmam a funcionalidade dos três genes na estirpe CPAC 15, com ênfase para o nopP, cuja funcionalidade em Bradyrhizobium japonicum foi descrita pela primeira vez.The objective of this work was to evaluate, by RT-qPCR, the expression of the nodC and nodW nodulation genes and of the nopP gene of the CPAC 15 strain, which probably play a role in the infection of soybean roots. Two experiments were done. In the first, the gene expression was evaluated in cells after incubation with genistein for 15 min, 1, 4 and 8 hours. Results showed that the three genes showed higher expression immediately after contact with the inducer (15 min. In the second experiment, the bacterium was grown in the presence of inducers (genistein or soybean seed exudates for 48 hours. The expression of the three genes was greater when induced by genistein, and the expression of nodC, nodW and nopP had higher values than the control. The results confirm the functionality of the three genes in the CPAC 15 strain, with an emphasis on the nopP, whose functionality in Bradyrhizobium japonicum was described for the first time.

Bone to pick: the importance of evaluating reference genes for RT-qPCR quantification of gene expression in craniosynostosis and bone-related tissues and cells

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Yang Xianxian

2012-05-01

Full Text Available Abstract Background RT-qPCR is a common tool for quantification of gene expression, but its accuracy is dependent on the choice and stability (steady state expression levels of the reference gene/s used for normalization. To date, in the bone field, there have been few studies to determine the most stable reference genes and, usually, RT-qPCR data is normalised to non-validated reference genes, most commonly GAPDH, ACTB and 18 S rRNA. Here we draw attention to the potential deleterious impact of using classical reference genes to normalise expression data for bone studies without prior validation of their stability. Results Using the geNorm and Normfinder programs, panels of mouse and human genes were assessed for their stability under three different experimental conditions: 1 disease progression of Crouzon syndrome (craniosynostosis in a mouse model, 2 proliferative culture of cranial suture cells isolated from craniosynostosis patients and 3 osteogenesis of a mouse bone marrow stromal cell line. We demonstrate that classical reference genes are not always the most ‘stable’ genes and that gene ‘stability’ is highly dependent on experimental conditions. Selected stable genes, individually or in combination, were then used to normalise osteocalcin and alkaline phosphatase gene expression data during cranial suture fusion in the craniosynostosis mouse model and strategies compared. Strikingly, the expression trends of alkaline phosphatase and osteocalcin varied significantly when normalised to the least stable, the most stable or the three most stable genes. Conclusion To minimise errors in evaluating gene expression levels, analysis of a reference panel and subsequent normalization to several stable genes is strongly recommended over normalization to a single gene. In particular, we conclude that use of single, non-validated “housekeeping” genes such as GAPDH, ACTB and 18 S rRNA, currently a widespread practice by researchers in

Quantitative Real-time PCR detection of putrescine-producing Gram-negative bacteria

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Kristýna Maršálková

2017-01-01

Full Text Available Biogenic amines are indispensable components of living cells; nevertheless these compounds could be toxic for human health in higher concentrations. Putrescine is supposed to be the major biogenic amine associated with microbial food spoilage. Development of reliable, fast and culture-independent molecular methods to detect bacteria producing biogenic amines deserves the attention, especially of the food industry in purpose to protect health. The objective of this study was to verify the newly designed primer sets for detection of two inducible genes adiA and speF together in Salmonella enterica and Escherichia coli genome by Real-time PCR. These forenamed genes encode enzymes in the metabolic pathway which leads to production of putrescine in Gram-negative bacteria. Moreover, relative expression of these genes was studied in E. coli CCM 3954 strain using Real-time PCR. In this study, sets of new primers for the detection two inducible genes (speF and adiA in Salmonella enterica and E. coli by Real-time PCR were designed and tested. Amplification efficiency of a Real-time PCR was calculated from the slope of the standard curves (adiA, speF, gapA. An efficiency in a range from 95 to 105 % for all tested reactions was achieved. The gene expression (R of adiA and speF genes in E. coli was varied depending on culture conditions. The highest gene expression of adiA and speF was observed at 6, 24 and 36 h (RadiA ~ 3, 5, 9; RspeF ~11, 10, 9; respectively after initiation of growth of this bacteria in nutrient broth medium enchired with amino acids. The results show that these primers could be used for relative quantification analysis of E. coli.

Expression study of CYP19A1 gene in a cohort of Iranian leiomyoma patients

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Leila Emrahi

2018-07-01

Full Text Available Background: CYP19A1 gene encodes aromatase, a microsomal key enzyme that catalyzes the synthesis of estrogens from androgens. Accumulating evidence has revealed that aromatase plays an important role in the pathogenesis of leiomyoma through increasing local concentration of estrogens. In this study, we examined the levels of CYP19A1 mRNA to determine the impact of aromatase overexpression in uterine leiomyoma growth. Subjects and methods: Tissues were obtained via myomectomy or hysterectomy from 30 patients. Total RNA was extracted and cDNA was synthesized from each frozen sample. Using SYBR Green dye, Real-time PCR assay was performed by sequence-specific primers. Relative mRNA expression was normalized to the mean of the Ct values determined for HPRT1. Gene expression ratio in each sample was determined relative to the mean ΔCt value of tumor-free margin samples. Results: PCR efficiencies for amplification reactions of HPRT1, and CYP19A genes were calculated as 0.93 and 0.96, respectively. Regression coefficients (R for standard curves were above 0.90. The obtained data revealed that the mean fold increase of CYP19A1 gene expression in leiomyoma samples relative to normal samples was 3.551 (95% CI: 0.04–6.64, S.E., 0.29–5.35. Conclusions: Our results were in accordance with previous studies and imply that up-regulation of CYP19A1 is correlated with the pathogenesis of leiomyoma tumors. We also observed that expression level of CYP19A1 was not linked to the tumor size or localization. It can be concluded that; up-regulation of aromatase is a key factor in the initiation of tumor development as well as tumor growth. Keywords: Leiomyoma, CYP19A1, Real-time PCR, Gene expression study

Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR

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Zou Ruiyang

2011-04-01

Full Text Available Abstract Background Accurate interpretation of quantitative PCR (qPCR data requires normalization using constitutively expressed reference genes. Ribosomal RNA is often used as a reference gene for transcriptional studies in E. coli. However, the choice of reliable reference genes has not been systematically validated. The objective of this study is to identify a set of reliable reference genes for transcription analysis in recombinant protein over-expression studies in E. coli. Results In this study, the meta-analysis of 240 sets of single-channel Affymetrix microarray data representing over-expressions of 63 distinct recombinant proteins in various E. coli strains identified twenty candidate reference genes that were stably expressed across all conditions. The expression of these twenty genes and two commonly used reference genes, rrsA encoding ribosomal RNA 16S and ihfB, was quantified by qPCR in E. coli cells over-expressing four genes of the 1-Deoxy-D-Xylulose 5-Phosphate pathway. From these results, two independent statistical algorithms identified three novel reference genes cysG, hcaT, and idnT but not rrsA and ihfB as highly invariant in two E. coli strains, across different growth temperatures and induction conditions. Transcriptomic data normalized by the geometric average of these three genes demonstrated that genes of the lycopene synthetic pathway maintained steady expression upon enzyme overexpression. In contrast, the use of rrsA or ihfB as reference genes led to the mis-interpretation that lycopene pathway genes were regulated during enzyme over-expression. Conclusion This study identified cysG/hcaT/idnT to be reliable novel reference genes for transcription analysis in recombinant protein producing E. coli.

Assessing gene expression during pathogenesis: Use of qRT-PCR to follow toxin production in the entomopathogenic fungus Beauveria bassiana during infection and immune response of the insect host Triatoma infestans.

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Lobo, Luciana S; Luz, Christian; Fernandes, Éverton K K; Juárez, M Patricia; Pedrini, Nicolás

2015-06-01

Entomopathogenic fungi secrete toxic secondary metabolites during the invasion of the insect hemocoel as part of the infection process. Although these compounds have been frequently mentioned as virulence factors, the roles of many of them remain poorly understood, including the question of whether they are expressed during the infection process. A major hurdle to this issue remains the low sensitivity of biochemical detection techniques (e.g., HPLC) within the complex samples that may contain trace quantities of fungal molecules inside the insect. In this study, quantitative reverse transcription real-time PCR (qRT-PCR) was used to measure the transcript levels within the insect fungal pathogen Beauveria bassiana, that encode for the synthetase enzymes of the secondary metabolites tenellin (BbtenS), beauvericin (BbbeaS) and bassianolide (BbbslS) during the infection of Triatoma infestans, a Chagas disease insect vector. Absolute quantification was performed at different time periods after insect treatment with various concentrations of propagules, either by immersing the insects in conidial suspensions or by injecting them with blastospores. Both BbtenS and BbbeaS were highly expressed in conidia-treated insects at days 3 and 12 post-treatment. In blastospore-injected insects, BbtenS and BbbeaS expression peaked at 24h post-injection and were also highly expressed in insect cadavers. The levels of BbbslS transcripts were much lower in all conditions tested. The expression patterns of insect genes encoding proteins that belong to the T. infestans humoral immune system were also evaluated with the same technique. This qPCR-based methodology can contribute to decifering the dynamics of entomopathogenic fungal infection at the molecular level. Copyright © 2015 Elsevier Inc. All rights reserved.

Environmental Regulation of Plant Gene Expression: An Rt-qPCR Laboratory Project for an Upper-Level Undergraduate Biochemistry or Molecular Biology Course

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Eickelberg, Garrett J.; Fisher, Alison J.

2013-01-01

We present a novel laboratory project employing "real-time" RT-qPCR to measure the effect of environment on the expression of the "FLOWERING LOCUS C" gene, a key regulator of floral timing in "Arabidopsis thaliana" plants. The project requires four 3-hr laboratory sessions and is aimed at upper-level undergraduate…

Defining suitable reference genes for RT-qPCR analysis on human sertoli cells after 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure.

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Ribeiro, Mariana Antunes; dos Reis, Mariana Bisarro; de Moraes, Leonardo Nazário; Briton-Jones, Christine; Rainho, Cláudia Aparecida; Scarano, Wellerson Rodrigo

2014-11-01

Quantitative real-time RT-PCR (qPCR) has proven to be a valuable molecular technique to quantify gene expression. There are few studies in the literature that describe suitable reference genes to normalize gene expression data. Studies of transcriptionally disruptive toxins, like tetrachlorodibenzo-p-dioxin (TCDD), require careful consideration of reference genes. The present study was designed to validate potential reference genes in human Sertoli cells after exposure to TCDD. 32 candidate reference genes were analyzed to determine their applicability. geNorm and NormFinder softwares were used to obtain an estimation of the expression stability of the 32 genes and to identify the most suitable genes for qPCR data normalization.

Selection of reference genes for qPCR in hairy root cultures of peanut

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Medrano Giuliana

2011-10-01

Full Text Available Abstract Background Hairy root cultures produced via Agrobacterium rhizogenes-mediated transformation have emerged as practical biological models to elucidate the biosynthesis of specialized metabolites. To effectively understand the expression patterns of the genes involved in the metabolic pathways of these compounds, reference genes need to be systematically validated under specific experimental conditions as established by the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines. In the present report we describe the first validation of reference genes for RT-qPCR in hairy root cultures of peanut which produce stilbenoids upon elicitor treatments. Results A total of 21 candidate reference genes were evaluated. Nineteen genes were selected based on previous qPCR studies in plants and two were from the T-DNAs transferred from A. rhizogenes. Nucleotide sequences of peanut candidate genes were obtained using their homologous sequences in Arabidopsis. To identify the suitable primers, calibration curves were obtained for each candidate reference gene. After data analysis, 12 candidate genes meeting standard efficiency criteria were selected. The expression stability of these genes was analyzed using geNorm and NormFinder algorithms and a ranking was established based on expression stability of the genes. Candidate reference gene expression was shown to have less variation in methyl jasmonate (MeJA treated root cultures than those treated with sodium acetate (NaOAc. Conclusions This work constitutes the first effort to validate reference genes for RT-qPCR in hairy roots. While these genes were selected under conditions of NaOAc and MeJA treatment, we anticipate these genes to provide good targets for reference genes for hairy roots under a variety of stress conditions. The lead reference genes were a gene encoding for a TATA box binding protein (TBP2 and a gene encoding a ribosomal protein (RPL8C. A

A PCR-aided transcript titration assay revealing very low expression of a gene at band 3p21 in 33 cells lines derived from all types of lung cancer

NARCIS (Netherlands)

Kok, K; Buchhagen, D L; Carritt, B; Buys, C H; van den Berg, Anke

1993-01-01

We have developed a general PCR-based method to quantify the amount of a specific mRNA present in a given cell line or tissue. We applied this quantitative PCR to analyse the expression of D8, a human gene which we recently identified in the chromosomal region 3p21, the common deletion region of

Reference gene identification for reliable normalisation of quantitative RT-PCR data in Setaria viridis.

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Nguyen, Duc Quan; Eamens, Andrew L; Grof, Christopher P L

2018-01-01

Quantitative real-time polymerase chain reaction (RT-qPCR) is the key platform for the quantitative analysis of gene expression in a wide range of experimental systems and conditions. However, the accuracy and reproducibility of gene expression quantification via RT-qPCR is entirely dependent on the identification of reliable reference genes for data normalisation. Green foxtail ( Setaria viridis ) has recently been proposed as a potential experimental model for the study of C 4 photosynthesis and is closely related to many economically important crop species of the Panicoideae subfamily of grasses, including Zea mays (maize), Sorghum bicolor (sorghum) and Sacchurum officinarum (sugarcane). Setaria viridis (Accession 10) possesses a number of key traits as an experimental model, namely; (i) a small sized, sequenced and well annotated genome; (ii) short stature and generation time; (iii) prolific seed production, and; (iv) is amendable to Agrobacterium tumefaciens -mediated transformation. There is currently however, a lack of reference gene expression information for Setaria viridis ( S. viridis ). We therefore aimed to identify a cohort of suitable S. viridis reference genes for accurate and reliable normalisation of S. viridis RT-qPCR expression data. Eleven putative candidate reference genes were identified and examined across thirteen different S. viridis tissues. Of these, the geNorm and NormFinder analysis software identified SERINE / THERONINE - PROTEIN PHOSPHATASE 2A ( PP2A ), 5 '- ADENYLYLSULFATE REDUCTASE 6 ( ASPR6 ) and DUAL SPECIFICITY PHOSPHATASE ( DUSP ) as the most suitable combination of reference genes for the accurate and reliable normalisation of S. viridis RT-qPCR expression data. To demonstrate the suitability of the three selected reference genes, PP2A , ASPR6 and DUSP , were used to normalise the expression of CINNAMYL ALCOHOL DEHYDROGENASE ( CAD ) genes across the same tissues. This approach readily demonstrated the suitably of the three

Differentially expressed genes of Tetrahymena thermophila in response to tributyltin (TBT) identified by suppression subtractive hybridization and real time quantitative PCR.

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Feng, Lifang; Miao, Wei; Wu, Yuxuan

2007-02-15

Tributyltin (TBT) is widely used as antifouling paints, agriculture biocides, and plastic stabilizers around the world, resulting in great pollution problem in aquatic environments. However, it has been short of the biomonitor to detect TBT in freshwater. We constructed the suppression subtractive hybridization library of Tetrahymena thermophila exposed to TBT, and screened out 101 Expressed Sequence Tags whose expressions were significantly up- or down-regulated with TBT treatment. From this, a series of genes related to the TBT toxicity were discovered, such as glutathione-S-transferase gene (down-regulated), plasma membrane Ca2+ ATPase isoforms 3 gene (up-regulated) and NgoA (up-regulated). Furthermore, their expressions under different concentrations of TBT treatment (0.5-40 ppb) were detected by real time fluorescent quantitative PCR. The differentially expressed genes of T. thermophila in response to TBT were identified, which provide the basic to make Tetrahymena as a sensitive, rapid and convenient TBT biomonitor in freshwater based on rDNA inducible expression system.

A multicenter study of viable PCR using propidium monoazide to detect Legionella in water samples.

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Scaturro, Maria; Fontana, Stefano; Dell'eva, Italo; Helfer, Fabrizia; Marchio, Michele; Stefanetti, Maria Vittoria; Cavallaro, Mario; Miglietta, Marilena; Montagna, Maria Teresa; De Giglio, Osvalda; Cuna, Teresa; Chetti, Leonarda; Sabattini, Maria Antonietta Bucci; Carlotti, Michela; Viggiani, Mariagabriella; Stenico, Alberta; Romanin, Elisa; Bonanni, Emma; Ottaviano, Claudio; Franzin, Laura; Avanzini, Claudio; Demarie, Valerio; Corbella, Marta; Cambieri, Patrizia; Marone, Piero; Rota, Maria Cristina; Bella, Antonino; Ricci, Maria Luisa

2016-07-01

Legionella quantification in environmental samples is overestimated by qPCR. Combination with a viable dye, such as Propidium monoazide (PMA), could make qPCR (named then vPCR) very reliable. In this multicentre study 717 artificial water samples, spiked with fixed concentrations of Legionella and interfering bacterial flora, were analysed by qPCR, vPCR and culture and data were compared by statistical analysis. A heat-treatment at 55 °C for 10 minutes was also performed to obtain viable and not-viable bacteria. When data of vPCR were compared with those of culture and qPCR, statistical analysis showed significant differences (P 0.05). Overall this study provided a good experimental reproducibility of vPCR but also highlighted limits of PMA in the discriminating capability of dead and live bacteria, making vPCR not completely reliable. Copyright © 2016 Elsevier Inc. All rights reserved.

Digital PCR assessment of MGMT promoter methylation coupled with reduced protein expression optimises prediction of response to alkylating agents in metastatic colorectal cancer patients.

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Sartore-Bianchi, Andrea; Pietrantonio, Filippo; Amatu, Alessio; Milione, Massimo; Cassingena, Andrea; Ghezzi, Silvia; Caporale, Marta; Berenato, Rosa; Falcomatà, Chiara; Pellegrinelli, Alessio; Bardelli, Alberto; Nichelatti, Michele; Tosi, Federica; De Braud, Filippo; Di Nicolantonio, Federica; Barault, Ludovic; Siena, Salvatore

2017-01-01

O(6)-methylguanine-DNA-methyltransferase (MGMT) is a repair protein, and its deficiency makes tumours more susceptible to the cytotoxic effect of alkylating agents. Five clinical trials with temozolomide or dacarbazine have been performed in metastatic colorectal cancer (mCRC) with selection based on methyl-specific PCR (MSP) testing with modest results. We hypothesised that mitigated results are consequences of unspecific patient selection and that alternative methodologies for MGMT testing such as immunohistochemistry (IHC) and digital polymerase chain reaction (PCR) could enhance patient enrolment. Formalin-fixed paraffin embedded archival tumour tissue samples from four phase II studies of temozolomide or dacarbazine in MGMT MSP-positive mCRCs were analysed by IHC for MGMT protein expression and by methyl-BEAMing (MB) for percentage of promoter methylation. Pooled data were then retrospectively analysed according to objective response rate, progression-free survival (PFS) and overall survival (OS). One hundred and five patients were included in the study. Twelve had achieved partial response (PR) (11.4%), 24 stable disease (SD; 22.9%) and 69 progressive disease (PD; 65.7%). Patients with PR/SD had lower IHC scores and higher MB levels than those with PD. MGMT expression by IHC was negatively and MB levels positively associated with PFS (p alkylating agents. Their combination could enhance patient selection in this setting. Copyright © 2016 Elsevier Ltd. All rights reserved.

A common base method for analysis of qPCR data and the application of simple blocking in qPCR experiments.

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Ganger, Michael T; Dietz, Geoffrey D; Ewing, Sarah J

2017-12-01

qPCR has established itself as the technique of choice for the quantification of gene expression. Procedures for conducting qPCR have received significant attention; however, more rigorous approaches to the statistical analysis of qPCR data are needed. Here we develop a mathematical model, termed the Common Base Method, for analysis of qPCR data based on threshold cycle values (C q ) and efficiencies of reactions (E). The Common Base Method keeps all calculations in the logscale as long as possible by working with log 10 (E) ∙ C q , which we call the efficiency-weighted C q value; subsequent statistical analyses are then applied in the logscale. We show how efficiency-weighted C q values may be analyzed using a simple paired or unpaired experimental design and develop blocking methods to help reduce unexplained variation. The Common Base Method has several advantages. It allows for the incorporation of well-specific efficiencies and multiple reference genes. The method does not necessitate the pairing of samples that must be performed using traditional analysis methods in order to calculate relative expression ratios. Our method is also simple enough to be implemented in any spreadsheet or statistical software without additional scripts or proprietary components.

Detection of adenoviruses in shellfish by means of conventional-PCR, nested-PCR, and integrated cell culture PCR (ICC/PCR).

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Rigotto, C; Sincero, T C M; Simões, C M O; Barardi, C R M

2005-01-01

We tested three PCR based methodologies to detect adenoviruses associated with cultivated oysters. Conventional-PCR, nested-PCR, and integrated cell culture-PCR (ICC/PCR) were first optimized using oysters seeded with know amounts of Adenovirus serotype 5 (Ad5). The maximum sensitivity for Ad5 detection was determined for each method, and then used to detect natural adenovirus contamination in oysters from three aquiculture farms in Florianopolis, Santa Catarina State, Brazil, over a period of 6 months. The results showed that the nested-PCR was more sensitive (limit of detection: 1.2 PFU/g of tissue) than conventional-PCR and ICC-PCR (limit of detection for both: 1.2 x 10(2)PFU/g of tissue) for detection of Ad5 in oyster extracts. Nested-PCR was able to detect 90% of Ad5 contamination in harvested oyster samples, while conventional-PCR was unable to detect Ad5 in any of the samples. The present work suggests that detection of human adenoviruses can be used as a tool to monitor the presence of human viruses in marine environments where shellfish grow, and that nested-PCR is the method of choice.

Identification of genes for normalization of real-time RT-PCR data in breast carcinomas

DEFF Research Database (Denmark)

Lyng, Maria B; Laenkholm, Anne-Vibeke; Pallisgaard, Niels

2008-01-01

BACKGROUND: Quantitative real-time RT-PCR (RT-qPCR) has become a valuable molecular technique in basic and translational biomedical research, and is emerging as an equally valuable clinical tool. Correlation of inter-sample values requires data normalization, which can be accomplished by various...... means, the most common of which is normalization to internal, stably expressed, reference genes. Recently, such traditionally utilized reference genes as GAPDH and B2M have been found to be regulated in various circumstances in different tissues, emphasizing the need to identify genes independent...... of factors influencing the tissue, and that are stably expressed within the experimental milieu. In this study, we identified genes for normalization of RT-qPCR data for invasive breast cancer (IBC), with special emphasis on estrogen receptor positive (ER+) IBC, but also examined their applicability to ER...

Gene expression profile and immunological evaluation of unique hypothetical unknown proteins of Mycobacterium leprae by using quantitative real-time PCR.

Science.gov (United States)

Kim, Hee Jin; Prithiviraj, Kalyani; Groathouse, Nathan; Brennan, Patrick J; Spencer, John S

2013-02-01

The cell-mediated immunity (CMI)-based in vitro gamma interferon release assay (IGRA) of Mycobacterium leprae-specific antigens has potential as a promising diagnostic means to detect those individuals in the early stages of M. leprae infection. Diagnosis of leprosy is a major obstacle toward ultimate disease control and has been compromised in the past by the lack of specific markers. Comparative bioinformatic analysis among mycobacterial genomes identified potential M. leprae-specific proteins called "hypothetical unknowns." Due to massive gene decay and the prevalence of pseudogenes, it is unclear whether any of these proteins are expressed or are immunologically relevant. In this study, we performed cDNA-based quantitative real-time PCR to investigate the expression status of 131 putative open reading frames (ORFs) encoding hypothetical unknowns. Twenty-six of the M. leprae-specific antigen candidates showed significant levels of gene expression compared to that of ESAT-6 (ML0049), which is an important T cell antigen of low abundance in M. leprae. Fifteen of 26 selected antigen candidates were expressed and purified in Escherichia coli. The seroreactivity to these proteins of pooled sera from lepromatous leprosy patients and cavitary tuberculosis patients revealed that 9 of 15 recombinant hypothetical unknowns elicited M. leprae-specific immune responses. These nine proteins may be good diagnostic reagents to improve both the sensitivity and specificity of detection of individuals with asymptomatic leprosy.

High-level expression of podoplanin in benign and malignant soft tissue tumors: immunohistochemical and quantitative real-time RT-PCR analysis.

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Xu, Yongjun; Ogose, Akira; Kawashima, Hiroyuki; Hotta, Tetsuo; Ariizumi, Takashi; Li, Guidong; Umezu, Hajime; Endo, Naoto

2011-03-01

Podoplanin is a 38 kDa mucin-type transmembrane glycoprotein that was first identified in rat glomerular epithelial cells (podocytes). It is expressed in normal lymphatic endothelium, but is absent from vascular endothelial cells. D2-40 is a commercially available mouse monoclonal antibody which binds to an epitope on human podoplanin. D2-40 immunoreactivity is therefore highly sensitive and specific for lymphatic endothelium. Recent investigations have shown widespread applications of immunohistochemical staining with D2-40 in evaluating podoplanin expression as an immunohistochemical marker for diagnosis and prognosis in various tumors. To determine whether the podoplanin (D2-40) antibody may be useful for the diagnosis of soft tissue tumors, 125 cases, including 4 kinds of benign tumors, 15 kinds of malignant tumors and 3 kinds of tumor-like lesions were immunostained using the D2-40 antibody. Total RNA was extracted from frozen tumor tissue obtained from 41 corresponding soft tissue tumor patients and 12 kinds of soft tissue tumor cell lines. Quantitative real-time PCR reactions were performed. Immunohistochemical and quantitative real-time RT-PCR analyses demonstrated the expression of the podoplanin protein and mRNA in the majority of benign and malignant soft tissue tumors and tumor-like lesions examined, with the exception of alveolar soft part sarcoma, embryonal and alveolar rhabdomyosarcoma, extraskeletal Ewing's sarcoma/peripheral primitive neuro-ectodermal tumor and lipoma, which were completely negative for podoplanin. Since it is widely and highly expressed in nearly all kinds of soft tissue tumors, especially in spindle cell sarcoma, myxoid type soft tissue tumors and soft tissue tumors of the nervous system, podoplanin is considered to have little value in the differential diagnosis of soft tissue tumors.

VEGF system expression by immunohistochemistry and real-time RT-PCR study on collared peccary placenta

DEFF Research Database (Denmark)

Santos, Tatiana C.; Oliveira, Moacir F.; Papa, Paula C.

2014-01-01

Vascular endothelial growth factor (VEGF) is known to induce endothelial cell proliferation, to promote cell migration, and to inhibit apoptosis, thus playing a central role in angiogenesis and in the regulation of vasculogenesis. The expression of the VEGF-ligand receptor system was studied in t...

A community resource for high-throughput quantitative RT-PCR analysis of transcription factor gene expression in Medicago truncatula

Directory of Open Access Journals (Sweden)

Redman Julia C

2008-07-01

Full Text Available Abstract Background Medicago truncatula is a model legume species that is currently the focus of an international genome sequencing effort. Although several different oligonucleotide and cDNA arrays have been produced for genome-wide transcript analysis of this species, intrinsic limitations in the sensitivity of hybridization-based technologies mean that transcripts of genes expressed at low-levels cannot be measured accurately with these tools. Amongst such genes are many encoding transcription factors (TFs, which are arguably the most important class of regulatory proteins. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR is the most sensitive method currently available for transcript quantification, and one that can be scaled up to analyze transcripts of thousands of genes in parallel. Thus, qRT-PCR is an ideal method to tackle the problem of TF transcript quantification in Medicago and other plants. Results We established a bioinformatics pipeline to identify putative TF genes in Medicago truncatula and to design gene-specific oligonucleotide primers for qRT-PCR analysis of TF transcripts. We validated the efficacy and gene-specificity of over 1000 TF primer pairs and utilized these to identify sets of organ-enhanced TF genes that may play important roles in organ development or differentiation in this species. This community resource will be developed further as more genome sequence becomes available, with the ultimate goal of producing validated, gene-specific primers for all Medicago TF genes. Conclusion High-throughput qRT-PCR using a 384-well plate format enables rapid, flexible, and sensitive quantification of all predicted Medicago transcription factor mRNAs. This resource has been utilized recently by several groups in Europe, Australia, and the USA, and we expect that it will become the 'gold-standard' for TF transcript profiling in Medicago truncatula.

Selecting and validating reference genes for quantitative real-time PCR in Plutella xylostella (L.).

Science.gov (United States)

You, Yanchun; Xie, Miao; Vasseur, Liette; You, Minsheng

2018-05-01

Gene expression analysis provides important clues regarding gene functions, and quantitative real-time PCR (qRT-PCR) is a widely used method in gene expression studies. Reference genes are essential for normalizing and accurately assessing gene expression. In the present study, 16 candidate reference genes (ACTB, CyPA, EF1-α, GAPDH, HSP90, NDPk, RPL13a, RPL18, RPL19, RPL32, RPL4, RPL8, RPS13, RPS4, α-TUB, and β-TUB) from Plutella xylostella were selected to evaluate gene expression stability across different experimental conditions using five statistical algorithms (geNorm, NormFinder, Delta Ct, BestKeeper, and RefFinder). The results suggest that different reference genes or combinations of reference genes are suitable for normalization in gene expression studies of P. xylostella according to the different developmental stages, strains, tissues, and insecticide treatments. Based on the given experimental sets, the most stable reference genes were RPS4 across different developmental stages, RPL8 across different strains and tissues, and EF1-α across different insecticide treatments. A comprehensive and systematic assessment of potential reference genes for gene expression normalization is essential for post-genomic functional research in P. xylostella, a notorious pest with worldwide distribution and a high capacity to adapt and develop resistance to insecticides.

WetLab-2: Providing Quantitative PCR Capabilities on ISS

Science.gov (United States)

Parra, Macarena; Jung, Jimmy Kar Chuen; Almeida, Eduardo; Boone, Travis David; Schonfeld, Julie; Tran, Luan Hoang

2015-01-01

The objective of NASA Ames Research Centers WetLab-2 Project is to place on the ISS a system capable of conducting gene expression analysis via quantitative real-time PCR (qRT-PCR) of biological specimens sampled or cultured on orbit. The WetLab-2 system is capable of processing sample types ranging from microbial cultures to animal tissues dissected on-orbit. The project has developed a RNA preparation module that can lyse cells and extract RNA of sufficient quality and quantity for use as templates in qRT-PCR reactions. Our protocol has the advantage that it uses non-toxic chemicals, alcohols or other organics. The resulting RNA is transferred into a pipette and then dispensed into reaction tubes that contain all lyophilized reagents needed to perform qRT-PCR reactions. These reaction tubes are mounted on rotors to centrifuge the liquid to the reaction window of the tube using a cordless drill. System operations require simple and limited crew actions including syringe pushes, valve turns and pipette dispenses. The resulting process takes less than 30 min to have tubes ready for loading into the qRT-PCR unit.The project has selected a Commercial-Off-The-Shelf (COTS) qRT-PCR unit, the Cepheid SmartCycler, that will fly in its COTS configuration. The SmartCycler has a number of advantages including modular design (16 independent PCR modules), low power consumption, rapid thermal ramp times and four-color detection. The ability to detect up to four fluorescent channels will enable multiplex assays that can be used to normalize for RNA concentration and integrity, and to study multiple genes of interest in each module. The WetLab-2 system will have the capability to downlink data from the ISS to the ground after a completed run and to uplink new programs. The ability to conduct qRT-PCR on-orbit eliminates the confounding effects on gene expression of reentry stresses and shock acting on live cells and organisms or the concern of RNA degradation of fixed samples. The

PCR-RFLP analyses for studying the diversity of GH and Pit-1 genes in Slovak Simmental cattle

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Anna Trakovická

2013-10-01

Full Text Available The aim of this study was evaluation of growth hormone (GH and specific pituitary transcription factor (Pit-1 genes diversity in population of 353 Slovak Simmental cows. The analyses were based on single nucleotide polymorphisms GH/AluI and Pit-1/HinfI detections. A polymorphic site of GH gene (AluI has been linked to differences in circulating metabolites, metabolic hormones and milk yield. Bovine Pit-1 is responsible for pituitary development and hormone secreting gene expression, including GH gene. The Pit-1/HinfI locus was associated with growth, milk production and reproduction performance in cattle. Samples of genomic DNA were analyzed by PCR-RFLP method. Digestion of GH gene PCR products with restriction enzyme AluI revealed allele L and V with frequency 0.695 and 0.305, respectively. The digested Pit-1 gene PCR products with enzyme HinfI revealed alleles A (0.249 and B (0.751. Dominant genotypes were for GH gene heterozygous LV (0.47 and for Pit-1 gene homozygous BB (0.56 animals. The observed heterozygosity, effective allele numbers and polymorphism information content of GH/AluI and Pit-1/HinfI bovine loci population were 0.42/0.37, 1.73/1.59 and 0.33/0.30, respectively. The median polymorphic information content of loci was also transferred to the higher observed homozygosity in population (0.58/0.63. Keywords: cattle, growth hormone, leptin, PCR, Pit-1, polymorphism.

Expression profiling identifies genes involved in emphysema severity

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Bowman Rayleen V

2009-09-01

Full Text Available Abstract Chronic obstructive pulmonary disease (COPD is a major public health problem. The aim of this study was to identify genes involved in emphysema severity in COPD patients. Gene expression profiling was performed on total RNA extracted from non-tumor lung tissue from 30 smokers with emphysema. Class comparison analysis based on gas transfer measurement was performed to identify differentially expressed genes. Genes were then selected for technical validation by quantitative reverse transcriptase-PCR (qRT-PCR if also represented on microarray platforms used in previously published emphysema studies. Genes technically validated advanced to tests of biological replication by qRT-PCR using an independent test set of 62 lung samples. Class comparison identified 98 differentially expressed genes (p p Gene expression profiling of lung from emphysema patients identified seven candidate genes associated with emphysema severity including COL6A3, SERPINF1, ZNHIT6, NEDD4, CDKN2A, NRN1 and GSTM3.

Fluorescence quantitative PCR in detection of HBV DNA

International Nuclear Information System (INIS)

Shen Zheng; Li Ming; Shen Xia

2003-01-01

Objective: To study the relationship between the serum content of HBV-DNA and expression of serological markers with HBV infection patients. Methods: Serum samples from 375 hepatitis B patients with different clinical status and 70 normal persons were quantitated for HBV-DNA by FQ-PCR. Results: The average of HBV-DNA contents in the patient in the groups of HBsAg (+) and of HBeAg(+) were significantly higher than those in the group of HBsAg(-) and of HBeAg(-). Even in the group of HBeAg negative, high HBV-DNA contents might still be present in both the HBeAg(+) and HBeAg(-) groups. Conclusion: FQ-PCR can be used to monitor the real state of HBV infection, replication and the course of disease

Validation of putative reference genes for normalization of Q-RT-PCR data from paraffin-embedded lymphoid tissue

DEFF Research Database (Denmark)

Green, Tina Marie; de Stricker, Karin; Møller, Michael Boe

2009-01-01

Normalization of quantitative reverse transcription-PCR (Q-RT-PCR) data to appropriate tissue-specific reference genes is an essential part of interpreting the results. This study aimed to determine the most appropriate reference genes for normalizing gene expressions in lymphatic tissue...... was 0.93 (Pnormalization with the appropriate reference genes. Thus, we show that formalin-fixed, paraffin-embedded lymphoid samples are suitable for Q-RT-PCR when using thoroughly validated reference genes....

Identification of Reference Genes for RT-qPCR Data Normalization in Cannabis sativa Stem Tissues

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Lauralie Mangeot-Peter

2016-09-01

Full Text Available Gene expression profiling via quantitative real-time PCR is a robust technique widely used in the life sciences to compare gene expression patterns in, e.g., different tissues, growth conditions, or after specific treatments. In the field of plant science, real-time PCR is the gold standard to study the dynamics of gene expression and is used to validate the results generated with high throughput techniques, e.g., RNA-Seq. An accurate relative quantification of gene expression relies on the identification of appropriate reference genes, that need to be determined for each experimental set-up used and plant tissue studied. Here, we identify suitable reference genes for expression profiling in stems of textile hemp (Cannabis sativa L., whose tissues (isolated bast fibres and core are characterized by remarkable differences in cell wall composition. We additionally validate the reference genes by analysing the expression of putative candidates involved in the non-oxidative phase of the pentose phosphate pathway and in the first step of the shikimate pathway. The goal is to describe the possible regulation pattern of some genes involved in the provision of the precursors needed for lignin biosynthesis in the different hemp stem tissues. The results here shown are useful to design future studies focused on gene expression analyses in hemp.

Identification of Reference Genes for RT-qPCR Data Normalization in Cannabis sativa Stem Tissues.

Science.gov (United States)

Mangeot-Peter, Lauralie; Legay, Sylvain; Hausman, Jean-Francois; Esposito, Sergio; Guerriero, Gea

2016-09-15

Gene expression profiling via quantitative real-time PCR is a robust technique widely used in the life sciences to compare gene expression patterns in, e.g., different tissues, growth conditions, or after specific treatments. In the field of plant science, real-time PCR is the gold standard to study the dynamics of gene expression and is used to validate the results generated with high throughput techniques, e.g., RNA-Seq. An accurate relative quantification of gene expression relies on the identification of appropriate reference genes, that need to be determined for each experimental set-up used and plant tissue studied. Here, we identify suitable reference genes for expression profiling in stems of textile hemp (Cannabis sativa L.), whose tissues (isolated bast fibres and core) are characterized by remarkable differences in cell wall composition. We additionally validate the reference genes by analysing the expression of putative candidates involved in the non-oxidative phase of the pentose phosphate pathway and in the first step of the shikimate pathway. The goal is to describe the possible regulation pattern of some genes involved in the provision of the precursors needed for lignin biosynthesis in the different hemp stem tissues. The results here shown are useful to design future studies focused on gene expression analyses in hemp.

Kolmogorov-Smirnov statistical test for analysis of ZAP-70 expression in B-CLL, compared with quantitative PCR and IgV(H) mutation status.

Science.gov (United States)

Van Bockstaele, Femke; Janssens, Ann; Piette, Anne; Callewaert, Filip; Pede, Valerie; Offner, Fritz; Verhasselt, Bruno; Philippé, Jan

2006-07-15

ZAP-70 has been proposed as a surrogate marker for immunoglobulin heavy-chain variable region (IgV(H)) mutation status, which is known as a prognostic marker in B-cell chronic lymphocytic leukemia (CLL). The flow cytometric analysis of ZAP-70 suffers from difficulties in standardization and interpretation. We applied the Kolmogorov-Smirnov (KS) statistical test to make analysis more straightforward. We examined ZAP-70 expression by flow cytometry in 53 patients with CLL. Analysis was performed as initially described by Crespo et al. (New England J Med 2003; 348:1764-1775) and alternatively by application of the KS statistical test comparing T cells with B cells. Receiver-operating-characteristics (ROC)-curve analyses were performed to determine the optimal cut-off values for ZAP-70 measured by the two approaches. ZAP-70 protein expression was compared with ZAP-70 mRNA expression measured by a quantitative PCR (qPCR) and with the IgV(H) mutation status. Both flow cytometric analyses correlated well with the molecular technique and proved to be of equal value in predicting the IgV(H) mutation status. Applying the KS test is reproducible, simple, straightforward, and overcomes a number of difficulties encountered in the Crespo-method. The KS statistical test is an essential part of the software delivered with modern routine analytical flow cytometers and is well suited for analysis of ZAP-70 expression in CLL. (c) 2006 International Society for Analytical Cytology.

Validation of cell-free culture using scanning electron microscopy (SEM) and gene expression studies.

Science.gov (United States)

Yang, R; Elankumaran, Y; Hijjawi, N; Ryan, U

2015-06-01

A cell-free culture system for Cryptosporidium parvum was analysed using scanning electron microscopy (SEM) to characterise life cycle stages and compare gene expression in cell-free culture and cell culture using HCT-8 cells. Cryptosporidium parvum samples were harvested at 2 h, 8 h, 14 h, 26 h, 50 h, 74 h, 98 h, 122 h and 170 h, chemically fixed and specimens were observed using a Zeiss 1555 scanning electron microscope. The presence of sporozoites, trophozoites and type I merozoites were identified by SEM. Gene expression in cell culture and cell-free culture was studied using reverse transcriptase quantitative PCR (RT-qPCR) of the sporozoite surface antigen protein (cp15), the glycoprotein 900 (gp900), the Cryptosporidium oocyst wall protein (COWP) and 18S ribosomal RNA (rRNA) genes in both cell free and conventional cell culture. In cell culture, cp15 expression peaked at 74 h, gp900 expression peaked at 74 h and 98 h and COWP expression peaked at 50 h. In cell-free culture, CP15 expression peaked at 98 h, gp900 expression peaked at 74 h and COWP expression peaked at 122 h. The present study is the first to compare gene expression of C. parvum in cell culture and cell-free culture and to characterise life cycle stages of C. parvum in cell-free culture using SEM. Findings from this study showed that gene expression patterns in cell culture and cell-free culture were similar but in cell-free culture, gene expression was delayed for CP15 and COWP in cell free culture compared with the cell culture system and was lower. Although three life cycle stageswere conclusively identified, improvements in SEM methodology should lead to the detection of more life cycle stages. Copyright © 2015 Elsevier Inc. All rights reserved.

Study of formation of green eggshell color in ducks through global gene expression.

Science.gov (United States)

Xu, Fa Qiong; Li, Ang; Lan, Jing Jing; Wang, Yue Ming; Yan, Mei Jiao; Lian, Sen Yang; Wu, Xu

2018-01-01

The green eggshell color produced by ducks is a threshold trait that can be influenced by various factors, such as hereditary, environment and nutrition. The aim of this study was to investigate the genetic regulation of the formation of eggs with green shells in Youxian ducks. We performed integrative analysis of mRNAs and miRNAs expression profiling in the shell gland samples from ducks by RNA-Seq. We found 124 differentially expressed genes that were associated with various pathways, such as the ATP-binding cassette (ABC) transporter and solute carrier supper family pathways. A total of 31 differentially expressed miRNAs were found between ducks laying green eggs and white eggs. KEGG pathway analysis of the predicted miRNA target genes also indicated the functional characteristics of these miRNAs; they were involved in the ABC transporter pathway and the solute carrier (SLC) supper family. Analysis with qRT-PCR was applied to validate the results of global gene expression, which showed a correlation between results obtained by RNA-seq and RT-qPCR. Moreover, a miRNA-mRNA interaction network was established using correlation analysis of differentially expressed mRNA and miRNA. Compared to ducks that lay white eggs, ducks that lay green eggs include six up-regulated miRNAs that had regulatory effects on 35 down-regulated genes, and seven down-regulated miRNAs which influenced 46 up-regulated genes. For example, the ABC transporter pathway could be regulated by expressing gga-miR-144-3p (up-regulated) with ABCG2 (up-regulated) and other miRNAs and genes. This study provides valuable information about mRNA and miRNA regulation in duck shell gland tissues, and provides foundational information for further study on the eggshell color formation and marker-assisted selection for Youxian duck breeding.

Study of formation of green eggshell color in ducks through global gene expression.

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Fa Qiong Xu

Full Text Available The green eggshell color produced by ducks is a threshold trait that can be influenced by various factors, such as hereditary, environment and nutrition. The aim of this study was to investigate the genetic regulation of the formation of eggs with green shells in Youxian ducks. We performed integrative analysis of mRNAs and miRNAs expression profiling in the shell gland samples from ducks by RNA-Seq. We found 124 differentially expressed genes that were associated with various pathways, such as the ATP-binding cassette (ABC transporter and solute carrier supper family pathways. A total of 31 differentially expressed miRNAs were found between ducks laying green eggs and white eggs. KEGG pathway analysis of the predicted miRNA target genes also indicated the functional characteristics of these miRNAs; they were involved in the ABC transporter pathway and the solute carrier (SLC supper family. Analysis with qRT-PCR was applied to validate the results of global gene expression, which showed a correlation between results obtained by RNA-seq and RT-qPCR. Moreover, a miRNA-mRNA interaction network was established using correlation analysis of differentially expressed mRNA and miRNA. Compared to ducks that lay white eggs, ducks that lay green eggs include six up-regulated miRNAs that had regulatory effects on 35 down-regulated genes, and seven down-regulated miRNAs which influenced 46 up-regulated genes. For example, the ABC transporter pathway could be regulated by expressing gga-miR-144-3p (up-regulated with ABCG2 (up-regulated and other miRNAs and genes. This study provides valuable information about mRNA and miRNA regulation in duck shell gland tissues, and provides foundational information for further study on the eggshell color formation and marker-assisted selection for Youxian duck breeding.

Quantitative real-time RT-PCR and chromogenic in situ hybridization

DEFF Research Database (Denmark)

Rosa, Fabíola E; Silveira, Sara M; Silveira, Cássia G T

2009-01-01

. METHODS: To elucidate the molecular profile of HER-2 status, mRNA and protein expression in 75 invasive breast carcinomas were analyzed by real time quantitative RT-PCR (qRT-PCR) and IHC, respectively. Amplifications were evaluated in 43 of these cases by CISH and in 11 by FISH. RESULTS: The concordance...

Applicability of integrated cell culture reverse transcriptase quantitative PCR (ICC-RTqPCR) for the simultaneous detection of the four human enteric enterovirus species in disinfection studies

Science.gov (United States)

A newly developed integrated cell culture reverse transcriptase quantitative PCR (ICC-RTqPCR) method and its applicability in UV disinfection studies is described. This method utilizes a singular cell culture system coupled with four RTqPCR assays to detect infectious serotypes t...

Reference Gene Screening for Analyzing Gene Expression Across Goat Tissue

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Yu Zhang

2013-12-01

Full Text Available Real-time quantitative PCR (qRT-PCR is one of the important methods for investigating the changes in mRNA expression levels in cells and tissues. Selection of the proper reference genes is very important when calibrating the results of real-time quantitative PCR. Studies on the selection of reference genes in goat tissues are limited, despite the economic importance of their meat and dairy products. We used real-time quantitative PCR to detect the expression levels of eight reference gene candidates (18S, TBP, HMBS, YWHAZ, ACTB, HPRT1, GAPDH and EEF1A2 in ten tissues types sourced from Boer goats. The optimal reference gene combination was selected according to the results determined by geNorm, NormFinder and Bestkeeper software packages. The analyses showed that tissue is an important variability factor in genes expression stability. When all tissues were considered, 18S, TBP and HMBS is the optimal reference combination for calibrating quantitative PCR analysis of gene expression from goat tissues. Dividing data set by tissues, ACTB was the most stable in stomach, small intestine and ovary, 18S in heart and spleen, HMBS in uterus and lung, TBP in liver, HPRT1 in kidney and GAPDH in muscle. Overall, this study provided valuable information about the goat reference genes that can be used in order to perform a proper normalisation when relative quantification by qRT-PCR studies is undertaken.

GENE EXPRESSION CHANGES AND ANTI-PROLIFERATIVE EFFECT OF NONI (Morinda citrifolia FRUIT EXTRACT ANALYSED BY REAL TIME-PCR

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hermansyah hermansyah

2017-05-01

Full Text Available To elucidate the anti-proliferative effect of noni (Morinda citrifolia fruit extract for a Saccharomyces cerevisiae model organism, analysis of gene expression changes related to cell cycle associated with inhibition effect of noni fruit extract was carried out. Anti-proliferative of noni fruit extract was analyzed using gene expression changes of Saccharomyces cerevisiae (strains FY833 and BY4741.  Transcriptional analysis of genes that play a role in cell cycle was conducted by growing cells on YPDAde broth medium containing 1% (w/v noni fruit extract, and then subjected using quantitative real-time polymerase chain reaction (RT-PCR.  Transcriptional level of genes CDC6 (Cell Division Cycle-6, CDC20 (Cell Division Cycle-20, FAR1 (Factor ARrest-1, FUS3 (FUSsion-3, SIC1 (Substrate/Subunit Inhibitor of Cyclin-dependent protein kinase-1, WHI5 (WHIskey-5, YOX1 (Yeast homeobOX-1 and YHP1 (Yeast Homeo-Protein-1 increased, oppositely genes expression of DBF4 (DumbBell Forming, MCM1 (Mini Chromosome Maintenance-1 and TAH11 (Topo-A Hypersensitive-11 decreased, while the expression level of genes CDC7 (Cell Division Cycle-7, MBP1 (MIul-box Binding Protein-1 and SWI6 (SWItching deficient-6 relatively unchanged. These results indicated that gene expression changes might associate with anti-proliferative effect from noni fruit extract. These gene expressions changes lead to the growth inhibition of S.cerevisiae cell because of cell cycle defect.

Splinkerette PCR for mapping transposable elements in Drosophila.

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Christopher J Potter

2010-04-01

Full Text Available Transposable elements (such as the P-element and piggyBac have been used to introduce thousands of transgenic constructs into the Drosophila genome. These transgenic constructs serve many roles, from assaying gene/cell function, to controlling chromosome arm rearrangement. Knowing the precise genomic insertion site for the transposable element is often desired. This enables identification of genomic enhancer regions trapped by an enhancer trap, identification of the gene mutated by a transposon insertion, or simplifying recombination experiments. The most commonly used transgene mapping method is inverse PCR (iPCR. Although usually effective, limitations with iPCR hinder its ability to isolate flanking genomic DNA in complex genomic loci, such as those that contain natural transposons. Here we report the adaptation of the splinkerette PCR (spPCR method for the isolation of flanking genomic DNA of any P-element or piggyBac. We report a simple and detailed protocol for spPCR. We use spPCR to 1 map a GAL4 enhancer trap located inside a natural transposon, pinpointing a master regulatory region for olfactory neuron expression in the brain; and 2 map all commonly used centromeric FRT insertion sites. The ease, efficiency, and efficacy of spPCR could make it a favored choice for the mapping of transposable element in Drosophila.

Validation of reference genes for normalization of qPCR mRNA expression levels in Staphylococcus aureus exposed to osmotic and lactic acid stress conditions encountered during food production and preservation.

Science.gov (United States)

Sihto, Henna-Maria; Tasara, Taurai; Stephan, Roger; Johler, Sophia

2014-07-01

Staphylococcus aureus represents the most prevalent cause of food-borne intoxications worldwide. While being repressed by competing bacteria in most matrices, this pathogen exhibits crucial competitive advantages during growth at high salt concentrations or low pH, conditions frequently encountered in food production and preservation. We aimed to identify reference genes that could be used to normalize qPCR mRNA expression levels during growth of S. aureus in food-related osmotic (NaCl) and acidic (lactic acid) stress adaptation models. Expression stability of nine housekeeping genes was evaluated in full (LB) and nutrient-deficient (CYGP w/o glucose) medium under conditions of osmotic (4.5% NaCl) and acidic stress (lactic acid, pH 6.0) after 2-h exposure. Among the set of candidate reference genes investigated, rplD, rpoB,gyrB, and rho were most stably expressed in LB and thus represent the most suitable reference genes for normalization of qPCR data in osmotic or lactic acid stress models in a rich medium. Under nutrient-deficient conditions, expression of rho and rpoB was highly stable across all tested conditions. The presented comprehensive data on changes in expression of various S. aureus housekeeping genes under conditions of osmotic and lactic acid stress facilitate selection of reference genes for qPCR-based stress response models. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Gene expression analysis in prostate cancer: the importance of the endogenous control.

LENUS (Irish Health Repository)

Vajda, Alice

2013-03-01

Aberrant gene expression is a hallmark of cancer. Quantitative reverse-transcription PCR (qRT-PCR) is the gold-standard for quantifying gene expression, and commonly employs a house-keeping gene (HKG) as an endogenous control to normalize results; the choice of which is critical for accurate data interpretation. Many factors, including sample type, pathological state, and oxygen levels influence gene expression including putative HKGs. The aim of this study was to determine the suitability of commonly used HKGs for qRT-PCR in prostate cancer.

Exploring valid reference genes for quantitative real-time PCR analysis in Sesamia inferens (Lepidoptera: Noctuidae.

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Meng Sun

Full Text Available The pink stem borer, Sesamia inferens, which is endemic in China and other parts of Asia, is a major pest of rice and causes significant yield loss in this host plant. Very few studies have addressed gene expression in S. inferens. Quantitative real-time PCR (qRT-PCR is currently the most accurate and sensitive method for gene expression analysis. In qRT-PCR, data are normalized using reference genes, which help control for internal differences and reduce error between samples. In this study, seven candidate reference genes, 18S ribosomal RNA (18S rRNA, elongation factor 1 (EF1, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, ribosomal protein S13 (RPS13, ribosomal protein S20 (RPS20, tubulin (TUB, and β-actin (ACTB were evaluated for their suitability in normalizing gene expression under different experimental conditions. The results indicated that three genes (RPS13, RPS20, and EF1 were optimal for normalizing gene expression in different insect tissues (head, epidermis, fat body, foregut, midgut, hindgut, Malpighian tubules, haemocytes, and salivary glands. 18S rRNA, EF1, and GAPDH were best for normalizing expression with respect to developmental stages and sex (egg masses; first, second, third, fourth, fifth, and sixth instar larvae; male and female pupae; and one-day-old male and female adults. 18S rRNA, RPS20, and TUB were optimal for fifth instars exposed to different temperatures (-8, -6, -4, -2, 0, and 27°C. To validate this recommendation, the expression profile of a target gene heat shock protein 83 gene (hsp83 was investigated, and results showed the selection was necessary and effective. In conclusion, this study describes reference gene sets that can be used to accurately measure gene expression in S. inferens.

Exploring valid reference genes for quantitative real-time PCR analysis in Sesamia inferens (Lepidoptera: Noctuidae).

Science.gov (United States)

Sun, Meng; Lu, Ming-Xing; Tang, Xiao-Tian; Du, Yu-Zhou

2015-01-01

The pink stem borer, Sesamia inferens, which is endemic in China and other parts of Asia, is a major pest of rice and causes significant yield loss in this host plant. Very few studies have addressed gene expression in S. inferens. Quantitative real-time PCR (qRT-PCR) is currently the most accurate and sensitive method for gene expression analysis. In qRT-PCR, data are normalized using reference genes, which help control for internal differences and reduce error between samples. In this study, seven candidate reference genes, 18S ribosomal RNA (18S rRNA), elongation factor 1 (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein S13 (RPS13), ribosomal protein S20 (RPS20), tubulin (TUB), and β-actin (ACTB) were evaluated for their suitability in normalizing gene expression under different experimental conditions. The results indicated that three genes (RPS13, RPS20, and EF1) were optimal for normalizing gene expression in different insect tissues (head, epidermis, fat body, foregut, midgut, hindgut, Malpighian tubules, haemocytes, and salivary glands). 18S rRNA, EF1, and GAPDH were best for normalizing expression with respect to developmental stages and sex (egg masses; first, second, third, fourth, fifth, and sixth instar larvae; male and female pupae; and one-day-old male and female adults). 18S rRNA, RPS20, and TUB were optimal for fifth instars exposed to different temperatures (-8, -6, -4, -2, 0, and 27°C). To validate this recommendation, the expression profile of a target gene heat shock protein 83 gene (hsp83) was investigated, and results showed the selection was necessary and effective. In conclusion, this study describes reference gene sets that can be used to accurately measure gene expression in S. inferens.

Using RNA-Seq Data to Evaluate Reference Genes Suitable for Gene Expression Studies in Soybean.

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Aldrin Kay-Yuen Yim

Full Text Available Differential gene expression profiles often provide important clues for gene functions. While reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR is an important tool, the validity of the results depends heavily on the choice of proper reference genes. In this study, we employed new and published RNA-sequencing (RNA-Seq datasets (26 sequencing libraries in total to evaluate reference genes reported in previous soybean studies. In silico PCR showed that 13 out of 37 previously reported primer sets have multiple targets, and 4 of them have amplicons with different sizes. Using a probabilistic approach, we identified new and improved candidate reference genes. We further performed 2 validation tests (with 26 RNA samples on 8 commonly used reference genes and 7 newly identified candidates, using RT-qPCR. In general, the new candidate reference genes exhibited more stable expression levels under the tested experimental conditions. The three newly identified candidate reference genes Bic-C2, F-box protein2, and VPS-like gave the best overall performance, together with the commonly used ELF1b. It is expected that the proposed probabilistic model could serve as an important tool to identify stable reference genes when more soybean RNA-Seq data from different growth stages and treatments are used.

Quantitative PCR and immunohistochemical analyses of HMGB1 and RAGE expression in canine disseminated histiocytic sarcoma (malignant histiocytosis).

Science.gov (United States)

Sterenczak, Katharina A; Kleinschmidt, Sven; Wefstaedt, Patrick; Eberle, Nina; Hewicker-Trautwein, Marion; Bullerdiek, Jörn; Nolte, Ingo; Murua Escobar, Hugo

2011-05-01

Disorders of histiocytic origin affecting humans and dogs share various similarities. Canine disseminated histiocytic sarcoma (DHS) (formerly known as malignant histiocytosis) is an aggressive neoplasm of interstitial dendritic cells (DCs). The receptor for glycation end products (RAGE) and the high mobility group box1 protein (HMGB1) have been shown to be required for the maturation and migration of DCs. Thus, deregulation of the expression of these genes could have a major effect on the progression of histiocytic disorders. Neoplastic canine DHS samples and non-neoplastic control samples were analysed immunohistochemically and via real-time PCR. Significant down-regulation of RAGE in the lung tumour samples and down-regulation of HMGB1 in the lung, lymph node and spleen tumour samples were detected compared to their non-neoplastic counterparts. RAGE and HMGB1 expression down-regulation in canine DHS points to a role in the progression of histiocytic disorders.

Selection of reliable reference genes for gene expression studies in Trichoderma afroharzianum LTR-2 under oxalic acid stress.

Science.gov (United States)

Lyu, Yuping; Wu, Xiaoqing; Ren, He; Zhou, Fangyuan; Zhou, Hongzi; Zhang, Xinjian; Yang, Hetong

2017-10-01

An appropriate reference gene is required to get reliable results from gene expression analysis by quantitative real-time reverse transcription PCR (qRT-PCR). In order to identify stable and reliable reference genes in Trichoderma afroharzianum under oxalic acid (OA) stress, six commonly used housekeeping genes, i.e., elongation factor 1, ubiquitin, ubiquitin-conjugating enzyme, glyceraldehyde-3-phosphate dehydrogenase, α-tubulin, actin, from the effective biocontrol isolate T. afroharzianum strain LTR-2 were tested for their expression during growth in liquid culture amended with OA. Four in silico programs (comparative ΔCt, NormFinder, geNorm and BestKeeper) were used to evaluate the expression stabilities of six candidate reference genes. The elongation factor 1 gene EF-1 was identified as the most stably expressed reference gene, and was used as the normalizer to quantify the expression level of the oxalate decarboxylase coding gene OXDC in T. afroharzianum strain LTR-2 under OA stress. The result showed that the expression of OXDC was significantly up-regulated as expected. This study provides an effective method to quantify expression changes of target genes in T. afroharzianum under OA stress. Copyright © 2017 Elsevier B.V. All rights reserved.

Wetlab-2 - Quantitative PCR Tools for Spaceflight Studies of Gene Expression Aboard the International Space Station

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Schonfeld, Julie E.

2015-01-01

Wetlab-2 is a research platform for conducting real-time quantitative gene expression analysis aboard the International Space Station. The system enables spaceflight genomic studies involving a wide variety of biospecimen types in the unique microgravity environment of space. Currently, gene expression analyses of space flown biospecimens must be conducted post flight after living cultures or frozen or chemically fixed samples are returned to Earth from the space station. Post-flight analysis is limited for several reasons. First, changes in gene expression can be transient, changing over a timescale of minutes. The delay between sampling on Earth can range from days to months, and RNA may degrade during this period of time, even in fixed or frozen samples. Second, living organisms that return to Earth may quickly re-adapt to terrestrial conditions. Third, forces exerted on samples during reentry and return to Earth may affect results. Lastly, follow up experiments designed in response to post-flight results must wait for a new flight opportunity to be tested.

Study the Three Extraction Methods for HBV DNA to Use in PCR

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N. Sheikh

2004-07-01

Full Text Available Diagnosis of Hepatitis B is important because of the its high prevalence. Recently PCR method , has found greater interest among different diagnostic methods. Several reports emphasis on some false negative results in those laboratories using PCR. The aim of this study was to compare three different procedures for HBV DNA extraction. A total 30 serum samples received from Shariati hospital. Sera was taken from patients having chronic Hepatitis with HBs antigen positive and HBe antigen negative. The sensitivity of guanidium hydrochloride method for extracting the HBV DNA from serum were evaluated and compared with phenol–chloroform and boiling methods. Diagnostic PCR kit was obtained from Cynagene contained taq polymerase, reaction mixture, dNTP, and buffer for reaction. A 353 bp product were amplified by amplification program provided in used PCR protocol. The comparison of results indicated that procedure was successful for amplification of the designed products from Hepatitis B in sera. Number of positive results were 16,19,23 and number of negative result were 14,11,7 for the boiling, phenol-chloroform and guanidium-hydrochloride extraction methods respectively.PCR method is the fastest diagnosis method and the most accurate procedure to identify Hepatitis B. Guanidium hydrochloride method was the most successful procedure studied in this survey for viruses.

In silico prediction and qPCR validation of novel sRNAs in Propionibacterium acnes KPA171202

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Praveen P. Balgir

2016-06-01

Full Text Available Propionibacterium acnes is an anaerobic, Gram-positive, opportunistic pathogen known to be involved in a wide variety of diseases ranging from mild acne to prostate cancer. Bacterial small non-coding RNAs are novel regulators of gene expression and are known to be involved in, virulence, pathogenesis, stress tolerance and adaptation to environmental changes in bacteria. The present study was undertaken keeping in view the lack of predicted sRNAs of P. acnes KPA171202 in databases. This report represents the first attempt to identify sRNAs in P. acnes KPA171202. A total of eight potential candidate sRNAs were predicted using SIPHT, one was found to have a Rfam homolog and seven were novel. Out of these seven predicted sRNAs, five were validated by reverse transcriptase-polymerase chain reaction (RT-PCR and sequencing. The expression of these sRNAs was quantified in different growth phases by qPCR (quantitative PCR. They were found to be expressed in both exponential and stationary stages of growth but with maximum expression in stationary phase which points to a regulatory role for them. Further investigation of their targets and regulatory functions is in progress.

Identification and validation of suitable endogenous reference genes for gene expression studies in human peripheral blood

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Turner Renee J

2009-08-01

Full Text Available Abstract Background Gene expression studies require appropriate normalization methods. One such method uses stably expressed reference genes. Since suitable reference genes appear to be unique for each tissue, we have identified an optimal set of the most stably expressed genes in human blood that can be used for normalization. Methods Whole-genome Affymetrix Human 2.0 Plus arrays were examined from 526 samples of males and females ages 2 to 78, including control subjects and patients with Tourette syndrome, stroke, migraine, muscular dystrophy, and autism. The top 100 most stably expressed genes with a broad range of expression levels were identified. To validate the best candidate genes, we performed quantitative RT-PCR on a subset of 10 genes (TRAP1, DECR1, FPGS, FARP1, MAPRE2, PEX16, GINS2, CRY2, CSNK1G2 and A4GALT, 4 commonly employed reference genes (GAPDH, ACTB, B2M and HMBS and PPIB, previously reported to be stably expressed in blood. Expression stability and ranking analysis were performed using GeNorm and NormFinder algorithms. Results Reference genes were ranked based on their expression stability and the minimum number of genes needed for nomalization as calculated using GeNorm showed that the fewest, most stably expressed genes needed for acurate normalization in RNA expression studies of human whole blood is a combination of TRAP1, FPGS, DECR1 and PPIB. We confirmed the ranking of the best candidate control genes by using an alternative algorithm (NormFinder. Conclusion The reference genes identified in this study are stably expressed in whole blood of humans of both genders with multiple disease conditions and ages 2 to 78. Importantly, they also have different functions within cells and thus should be expressed independently of each other. These genes should be useful as normalization genes for microarray and RT-PCR whole blood studies of human physiology, metabolism and disease.

Evaluation of suitable reference genes for gene expression studies in bovine muscular tissue

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Dunner Susana

2008-09-01

Full Text Available Abstract Background Real-time reverse transcriptase quantitative polymerase chain reaction (real-time RTqPCR is a technique used to measure mRNA species copy number as a way to determine key genes involved in different biological processes. However, the expression level of these key genes may vary among tissues or cells not only as a consequence of differential expression but also due to different factors, including choice of reference genes to normalize the expression levels of the target genes; thus the selection of reference genes is critical for expression studies. For this purpose, ten candidate reference genes were investigated in bovine muscular tissue. Results The value of stability of ten candidate reference genes included in three groups was estimated: the so called 'classical housekeeping' genes (18S, GAPDH and ACTB, a second set of genes used in expression studies conducted on other tissues (B2M, RPII, UBC and HMBS and a third set of novel genes (SF3A1, EEF1A2 and CASC3. Three different statistical algorithms were used to rank the genes by their stability measures as produced by geNorm, NormFinder and Bestkeeper. The three methods tend to agree on the most stably expressed genes and the least in muscular tissue. EEF1A2 and HMBS followed by SF3A1, ACTB, and CASC3 can be considered as stable reference genes, and B2M, RPII, UBC and GAPDH would not be appropriate. Although the rRNA-18S stability measure seems to be within the range of acceptance, its use is not recommended because its synthesis regulation is not representative of mRNA levels. Conclusion Based on geNorm algorithm, we propose the use of three genes SF3A1, EEF1A2 and HMBS as references for normalization of real-time RTqPCR in muscle expression studies.

Expression studies of the obesity candidate gene FTO in pig

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Madsen, Majbritt Busk; Birck, Malene Muusfeldt; Fredholm, Merete

2010-01-01

Obesity is an increasing problem worldwide and research on candidate genes in good animal models is highly needed. The pig is an excellent model as its metabolism, organ size, and eating habits resemble that of humans. The present study is focused on the characterization of the fat mass and obesity...... associated gene (FTO) in pig. This gene has recently been associated with increased body mass index in several human populations. To establish information on the expression profile of FTO in the pig we performed quantitative PCR in a panel of adult pig tissues and in tissues sampled at different...... and cerebellum). Additionally, in order to see the involvement of the FTO gene in obesity, the changes in expression level were investigated in a nutritional study in brain of Gottingen minipigs under a high cholesterol diet. Significantly higher (P

Validation of reference genes for quantitative real-time PCR in Périgord black truffle (Tuber melanosporum) developmental stages.

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Zarivi, Osvaldo; Cesare, Patrizia; Ragnelli, Anna Maria; Aimola, Pierpaolo; Leonardi, Marco; Bonfigli, Antonella; Colafarina, Sabrina; Poma, Anna Maria; Miranda, Michele; Pacioni, Giovanni

2015-08-01

The symbiotic fungus Tuber melanosporum Vittad. (Périgord black truffle) belongs to the Ascomycota and forms mutualistic symbiosis with tree and shrub roots. This truffle has a high value in a global market and is cultivated in many countries of both hemispheres. The publication of the T. melanosporum genome has given researchers unique opportunities to learn more about the biology of the fungus. Real-time quantitative PCR (qRT-PCR) is a definitive technique for quantitating differences in transcriptional gene expression levels between samples. To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable housekeeping genes is required. These housekeeping genes must show stable expression under given experimental conditions for the qRT-PCR results to be accurate. Unfortunately, there are no studies on the stability of housekeeping genes used in T. melanosporum development. In this study, we present a morphological and microscopical classification of the developmental stages of T. melanosporum fruit body, and investigate the expression levels of 12 candidate reference genes (18S rRNA; 5.8S rRNA; Elongation factor 1-alpha; Elongation factor 1-beta; α-tubulin; 60S ribosomal protein L29; β-tubulin; 40S ribosomal protein S1; 40S ribosomal protein S3; Glucose-6-phosphate dehydrogenase; β-actin; Ubiquitin-conjugating enzyme). To evaluate the suitability of these genes as endogenous controls, five software-based approaches and one web-based comprehensive tool (RefFinder) were used to analyze and rank the tested genes. We demonstrate here that the 18S rRNA gene shows the most stable expression during T. melanosporum development and that a set of three genes, 18S rRNA, Elongation factor 1-alpha and 40S ribosomal protein S3, is the most suitable to normalize qRT-PCR data from all the analyzed developmental stages; conversely, 18S rRNA, Glucose-6-phosphate dehydrogenase and Elongation factor 1-alpha are the most suitable

Selection of Reference Genes for Expression Study in Pulp and Seeds of Theobroma grandiflorum (Willd. ex Spreng. Schum.

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Lucas Ferraz Dos Santos

Full Text Available Cupuassu (Theobroma grandiflorum [Willd. ex Spreng.] Schum is a species of high economic importance in Brazil with great potential at international level due to the multiple uses of both its seeds and pulp in the industry of sweets and cosmetics. For this reason, the cupuassu breeding program focused on the selection of genotypes with high pulp and seed quality-selection associated with the understanding of the mechanisms involved in fruit formation. Gene expression is one of the most used approaches related to such understanding. In this sense, quantitative real-time PCR (qPCR is a powerful tool, since it rapidly and reliably quantifies gene expression levels across different experimental conditions. The analysis by qPCR and the correct interpretation of data depend on signal normalization using reference genes, i.e. genes presenting a uniform pattern of expression in the analyzed samples. Here, we selected and analyzed the expression of five genes from cupuassu (ACP, ACT, GAPDH, MDH, TUB to be used as candidates for reference genes on pulp and seed of young, maturing and mature cupuassu fruits. The evaluation of the gene expression stability was obtained using the NormFinder, geNorm and BestKeeper programs. In general, our results indicated that the GAPDH and MDH genes constituted the best combination as reference genes to analyze the expression of cupuassu samples. To our knowledge, this is the first report of reference gene definition in cupuassu, and these results will support subsequent analysis related to gene expression studies in cupuassu plants subjected to different biotic or abiotic conditions as well as serve as a tool for diversity analysis based on pulp and seed quality.

PCR melting profile (PCR MP - a new tool for differentiation of Candida albicans strains

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Nowak Magdalena

2009-11-01

Full Text Available Abstract Background We have previously reported the use of PCR Melting Profile (PCR MP technique based on using low denaturation temperatures during ligation mediated PCR (LM PCR for bacterial strain differentiation. The aim of the current study was to evaluate this method for intra-species differentiation of Candida albicans strains. Methods In total 123 Candida albicans strains (including 7 reference, 11 clinical unrelated, and 105 isolates from patients of two hospitals in Poland were examined using three genotyping methods: PCR MP, macrorestriction analysis of the chromosomal DNA by pulsed-field gel electrophoresis (REA-PFGE and RAPD techniques. Results The genotyping results of the PCR MP were compared with results from REA-PFGE and RAPD techniques giving 27, 26 and 25 unique types, respectively. The results showed that the PCR MP technique has at least the same discriminatory power as REA-PFGE and RAPD. Conclusion Data presented here show for the first time the evaluation of PCR MP technique for candidial strains differentiation and we propose that this can be used as a relatively simple and cheap technique for epidemiological studies in short period of time in hospital.

Gene Expression Profiling in Fish Toxicology: A Review.

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Kumar, Girish; Denslow, Nancy D

In this review, we present an overview of transcriptomic responses to chemical exposures in a variety of fish species. We have discussed the use of several molecular approaches such as northern blotting, differential display reverse transcription-polymerase chain reaction (DDRT-PCR), suppression subtractive hybridization (SSH), real time quantitative PCR (RT-qPCR), microarrays, and next-generation sequencing (NGS) for measuring gene expression. These techniques have been mainly used to measure the toxic effects of single compounds or simple mixtures in laboratory conditions. In addition, only few studies have been conducted to examine the biological significance of differentially expressed gene sets following chemical exposure. Therefore, future studies should focus more under field conditions using a multidisciplinary approach (genomics, proteomics and metabolomics) to understand the synergetic effects of multiple environmental stressors and to determine the functional significance of differentially expressed genes. Nevertheless, recent developments in NGS technologies and decreasing costs of sequencing holds the promise to uncover the complexity of anthropogenic impacts and biological effects in wild fish populations.

Identification of reference genes for RT-qPCR in ovine mammary tissue during late pregnancy and lactation and in response to maternal nutritional programming.

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Paten, A M; Pain, S J; Peterson, S W; Blair, H T; Kenyon, P R; Dearden, P K; Duncan, E J

2014-08-01

The mammary gland is a complex tissue consisting of multiple cell types which, over the lifetime of an animal, go through repeated cycles of development associated with pregnancy, lactation and involution. The mammary gland is also known to be sensitive to maternal programming by environmental stimuli such as nutrition. The molecular basis of these adaptations is of significant interest, but requires robust methods to measure gene expression. Reverse-transcription quantitative PCR (RT-qPCR) is commonly used to measure gene expression, and is currently the method of choice for validating genome-wide expression studies. RT-qPCR requires the selection of reference genes that are stably expressed over physiological states and treatments. In this study we identify suitable reference genes to normalize RT-qPCR data for the ovine mammary gland in two physiological states; late pregnancy and lactation. Biopsies were collected from offspring of ewes that had been subjected to different nutritional paradigms during pregnancy to examine effects of maternal programming on the mammary gland of the offspring. We evaluated eight candidate reference genes and found that two reference genes (PRPF3 and CUL1) are required for normalising RT-qPCR data from pooled RNA samples, but five reference genes are required for analyzing gene expression in individual animals (SENP2, EIF6, MRPL39, ATP1A1, CUL1). Using these stable reference genes, we showed that TET1, a key regulator of DNA methylation, is responsive to maternal programming and physiological state. The identification of these novel reference genes will be of utility to future studies of gene expression in the ovine mammary gland. Copyright © 2014 the American Physiological Society.

Pathway-focused PCR array profiling of enriched populations of laser capture microdissected hippocampal cells after traumatic brain injury.

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Deborah R Boone

Full Text Available Cognitive deficits in survivors of traumatic brain injury (TBI are associated with irreversible neurodegeneration in brain regions such as the hippocampus. Comparative gene expression analysis of dying and surviving neurons could provide insight into potential therapeutic targets. We used two pathway-specific PCR arrays (RT2 Profiler Apoptosis and Neurotrophins & Receptors PCR arrays to identify and validate TBI-induced gene expression in dying (Fluoro-Jade-positive or surviving (Fluoro-Jade-negative pyramidal neurons obtained by laser capture microdissection (LCM. In the Apoptosis PCR array, dying neurons showed significant increases in expression of genes associated with cell death, inflammation, and endoplasmic reticulum (ER stress compared with adjacent, surviving neurons. Pro-survival genes with pleiotropic functions were also significantly increased in dying neurons compared to surviving neurons, suggesting that even irreversibly injured neurons are able to mount a protective response. In the Neurotrophins & Receptors PCR array, which consists of genes that are normally expected to be expressed in both groups of hippocampal neurons, only a few genes were expressed at significantly different levels between dying and surviving neurons. Immunohistochemical analysis of selected, differentially expressed proteins supported the gene expression data. This is the first demonstration of pathway-focused PCR array profiling of identified populations of dying and surviving neurons in the brain after TBI. Combining precise laser microdissection of identifiable cells with pathway-focused PCR array analysis is a practical, low-cost alternative to microarrays that provided insight into neuroprotective signals that could be therapeutically targeted to ameliorate TBI-induced neurodegeneration.

High-Density Real-Time PCR-Based in Vivo Toxicogenomic Screen to Predict Organ-Specific Toxicity

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Laszlo G. Puskas

2011-09-01

Full Text Available Toxicogenomics, based on the temporal effects of drugs on gene expression, is able to predict toxic effects earlier than traditional technologies by analyzing changes in genomic biomarkers that could precede subsequent protein translation and initiation of histological organ damage. In the present study our objective was to extend in vivo toxicogenomic screening from analyzing one or a few tissues to multiple organs, including heart, kidney, brain, liver and spleen. Nanocapillary quantitative real-time PCR (QRT-PCR was used in the study, due to its higher throughput, sensitivity and reproducibility, and larger dynamic range compared to DNA microarray technologies. Based on previous data, 56 gene markers were selected coding for proteins with different functions, such as proteins for acute phase response, inflammation, oxidative stress, metabolic processes, heat-shock response, cell cycle/apoptosis regulation and enzymes which are involved in detoxification. Some of the marker genes are specific to certain organs, and some of them are general indicators of toxicity in multiple organs. Utility of the nanocapillary QRT-PCR platform was demonstrated by screening different references, as well as discovery of drug-like compounds for their gene expression profiles in different organs of treated mice in an acute experiment. For each compound, 896 QRT-PCR were done: four organs were used from each of the treated four animals to monitor the relative expression of 56 genes. Based on expression data of the discovery gene set of toxicology biomarkers the cardio- and nephrotoxicity of doxorubicin and sulfasalazin, the hepato- and nephrotoxicity of rotenone, dihydrocoumarin and aniline, and the liver toxicity of 2,4-diaminotoluene could be confirmed. The acute heart and kidney toxicity of the active metabolite SN-38 from its less toxic prodrug, irinotecan could be differentiated, and two novel gene markers for hormone replacement therapy were identified

Cloning and evaluation of reference genes for quantitative real-time PCR analysis in Amorphophallus

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Kai Wang

2017-04-01

Full Text Available Quantitative real-time reverse transcription PCR (RT-qPCR has been widely used in the detection and quantification of gene expression levels because of its high accuracy, sensitivity, and reproducibility as well as its large dynamic range. However, the reliability and accuracy of RT-qPCR depends on accurate transcript normalization using stably expressed reference genes. Amorphophallus is a perennial plant with a high content of konjac glucomannan (KGM in its corm. This crop has been used as a food source and as a traditional medicine for thousands of years. Without adequate knowledge of gene expression profiles, there has been no report of validated reference genes in Amorphophallus. In this study, nine genes that are usually used as reference genes in other crops were selected as candidate reference genes. These putative sequences of these genes Amorphophallus were cloned by the use of degenerate primers. The expression stability of each gene was assessed in different tissues and under two abiotic stresses (heat and waterlogging in A. albus and A. konjac. Three distinct algorithms were used to evaluate the expression stability of the candidate reference genes. The results demonstrated that EF1-a, EIF4A, H3 and UBQ were the best reference genes under heat stress in Amorphophallus. Furthermore, EF1-a, EIF4A, TUB, and RP were the best reference genes in waterlogged conditions. By comparing different tissues from all samples, we determined that EF1-α, EIF4A, and CYP were stable in these sets. In addition, the suitability of these reference genes was confirmed by validating the expression of a gene encoding the small heat shock protein SHSP, which is related to heat stress in Amorphophallus. In sum, EF1-α and EIF4A were the two best reference genes for normalizing mRNA levels in different tissues and under various stress treatments, and we suggest using one of these genes in combination with 1 or 2 reference genes associated with different

Análise de citocinas pela RT-PCR em pacientes com rinite alérgica RT-PCR cytokine study in patients with allergic rhinitis

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Tarcimara Moreira da Silva

2009-02-01

Full Text Available Rinite alérgica é uma doença que decorre de um processo inflamatório da mucosa nasal conseqüente à reação de hipersensibilidade a alérgenos inalatórios e, eventualmente, alimentares. É mediada por IgE, envolvendo diferentes células, mediadores e citocinas. OBJETIVO: Avaliar as transcrições para as seguintes citocinas: IL-4, IL-5, IL-8 e IFN-gama, particularmente importantes no processo alérgico nasal, principalmente IL-4 e IL-5. Neste estudo, optou-se por avaliar os pacientes atópicos fora das crises alérgicas, com a finalidade de se conhecer as expressões das citocinas neste período. MATERIAL E MÉTODO: Realizou-se um estudo transversal e prospectivo, selecionando-se 30 pacientes, sendo 13 pacientes portadores de rinite alérgica paucissintomáticos e 17 pacientes não-atópicos. Os grupos foram selecionados através da história, do exame clínico otorrinolaringológico e do teste alérgico cutâneo - Prick Test. O perfil das citocinas foi pesquisado nos fragmentos de mucosa nasal, através da RT-PCR semiquantitativa, escolhida por apresentar boa reprodutibilidade e especificidade, utilizando-se como referência o gene da Beta-actina. RESULTADOS: Os valores de IL-5, IL-8, IFN-gama mantiveram-se homogêneos em relação ao grupo controle. A IL-4 apresentou diferença com significância estatística. CONCLUSÃO: Os pacientes alérgicos paucissintomáticos apresentaram normalização da expressão das citocinas na mucosa nasal à exceção de IL-4.Allergic rhinitis is an inflammatory reaction of the nasal mucosa, in consequence of an IgE mediated hypersensitive reaction to inhaling allergens, involving different mediators and cytokine cells. AIM: The purpose of this study was to evaluate the transcriptions for IL-4, IL-5, IL-8 and IFN-gama, particularly important in the nasal allergy process, especially IL-4 and IL-5. For this study we decided to evaluate atopic patients who were free from allergic crises, with the purpose of

Measurement of indicator genes using global complementary DNA (cDNA) amplification, by polyadenylic acid reverse transcriptase polymerase chain reaction (poly A RT-PCR): A feasibility study using paired samples from tissue and ductal juice in patients undergoing pancreatoduodenectomy.

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Sanyal, Sudip; Siriwardena, Ajith K; Byers, Richard

2018-06-01

The aim of this study is to compare gene expression profiles in RNA isolated from pancreatic ductal juice with the RNA expression profiles of the same genes from matched intra-operative tissue samples from pancreatic tumours. Intra-operative sampling of pancreatic juice and collection of matched tissue samples was undertaken in patients undergoing pancreatoduodenectomy for clinically suspected pancreatic cancer and a precursor lesion, main-duct intraductal papillary mucinous neoplasm. RNA was isolated and Poly A PCR was used to globally amplify the RNA. Real-time polymerase chain reaction (RT-PCR) was used to measure expression levels of 17 genes selected from microarray studies. Spearman's rank correlation test was used to examine the relationship of gene expression between pancreatic juice and tissue. The study was approved by Regional Ethics Committee. Mesothelin (MSLN) showed significant correlation (p cDNA using poly A PCR is technically feasible. Application of the technique to non-invasively obtained pancreatic juice during endoscopic assessment of tumours and the use of gene arrays of cancer indicator genes are the next steps in development of this technique. Copyright © 2018 IAP and EPC. Published by Elsevier B.V. All rights reserved.

Longitudinal monitoring of bottlenose dolphin leukocyte cytokine mRNA responsiveness by qPCR

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Both veterinarians caring for bottlenose dolphins (Tursiops truncatus) in managed populations and researchers monitoring wild populations use blood-based diagnostics to monitor bottlenose dolphin health. Quantitative PCR (qPCR) can be used to assess cytokine expression patterns of peripheral blood m...

Exploring Valid Reference Genes for Quantitative Real-time PCR Analysis in Plutella xylostella (Lepidoptera: Plutellidae)

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Fu, Wei; Xie, Wen; Zhang, Zhuo; Wang, Shaoli; Wu, Qingjun; Liu, Yong; Zhou, Xiaomao; Zhou, Xuguo; Zhang, Youjun

2013-01-01

Abstract: Quantitative real-time PCR (qRT-PCR), a primary tool in gene expression analysis, requires an appropriate normalization strategy to control for variation among samples. The best option is to compare the mRNA level of a target gene with that of reference gene(s) whose expression level is stable across various experimental conditions. In this study, expression profiles of eight candidate reference genes from the diamondback moth, Plutella xylostella, were evaluated under diverse experimental conditions. RefFinder, a web-based analysis tool, integrates four major computational programs including geNorm, Normfinder, BestKeeper, and the comparative ΔCt method to comprehensively rank the tested candidate genes. Elongation factor 1 (EF1) was the most suited reference gene for the biotic factors (development stage, tissue, and strain). In contrast, although appropriate reference gene(s) do exist for several abiotic factors (temperature, photoperiod, insecticide, and mechanical injury), we were not able to identify a single universal reference gene. Nevertheless, a suite of candidate reference genes were specifically recommended for selected experimental conditions. Our finding is the first step toward establishing a standardized qRT-PCR analysis of this agriculturally important insect pest. PMID:23983612

Multiplex reverse transcription polymerase chain reaction to study the expression of virulence and stress response genes in Staphylococcus aureus.

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Shrihari, Rohinishree Yadahalli; Singh, Negi Pradeep

2012-02-01

Staphylococcus aureus survives well in different stress conditions. The ability of this organism to adapt to various stresses is the result of a complex regulatory response, which is attributed to regulation of multiple genes. The aims of the present study were (1) to develop a multiplex PCR for the detection of genes which are involved in stress adaptation (asp23, dnaK, and groEL); alternative sigma factor (sigB) and virulence determination (entB and spa) and (2) to study the expression of these genes during stress conditions for S. aureus culture collection strains (FRI 722 and ATCC 6538) and S. aureus food isolates at mRNA level using multiplex reverse transcription polymerase chain reaction (RT-PCR). During heat shock treatment groEL, dnaK, asp23, sodA, entB, spa, and sigB genes were up regulated up to 2.58, 2.07, 2.76, 2.55, 3.55, 2.71, and 2.62- folds, respectively, whereas in acid shock treatment, sodA and groEL were up regulated; dnaK was downregulated; and entB and sigB genes were not expressed in food isolates. Multiplex PCR assay standardized in this study offers an inexpensive alternative to uniplex PCR for detection of various virulence and stress response genes. This study is relevant to rapid and accurate detection of potential pathogenic S. aureus in foods. © 2012 Institute of Food Technologists®

Validation of Suitable Reference Genes for RT-qPCR Data in Achyranthes bidentata Blume under Different Experimental Conditions

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Jinting Li

2017-05-01

Full Text Available Real-time quantitative polymerase chain reaction (RT-qPCR is a sensitive technique for gene expression studies. However, choosing the appropriate reference gene is essential to obtain reliable results for RT-qPCR assays. In the present work, the expression of eight candidate reference genes, EF1-α (elongation factor 1-α, GAPDH (glyceraldehyde 3-phosphate dehydrogenase, UBC (ubiquitin-conjugating enzyme, UBQ (polyubiquitin, ACT (actin, β-TUB (β-tubulin, APT1 (adenine phosphoribosyltransferase 1, and 18S rRNA (18S ribosomal RNA, was evaluated in Achyranthes bidentata samples using two algorithms, geNorm and NormFinder. The samples were classified into groups according to developmental stages, various tissues, stresses (cold, heat, drought, NaCl, and hormone treatments (MeJA, IBA, SA. Suitable combination of reference genes for RT-qPCR normalization should be applied according to different experimental conditions. In this study, EF1-α, UBC, and ACT genes were verified as the suitable reference genes across all tested samples. To validate the suitability of the reference genes, we evaluated the relative expression of CAS, which is a gene that may be involved in phytosterol synthesis. Our results provide the foundation for gene expression analysis in A. bidentata and other species of Amaranthaceae.

Identification of circulating miRNA biomarkers based on global quantitative real-time PCR profiling

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Kang Kang

2012-02-01

Full Text Available Abstract MicroRNAs (miRNAs are small noncoding RNAs (18-25 nucleotides that regulate gene expression at the post-transcriptional level. Recent studies have demonstrated the presence of miRNAs in the blood circulation. Deregulation of miRNAs in serum or plasma has been associated with many diseases including cancers and cardiovascular diseases, suggesting the possible use of miRNAs as diagnostic biomarkers. However, the detection of the small amount of miRNAs found in serum or plasma requires a method with high sensitivity and accuracy. Therefore, the current study describes polymerase chain reaction (PCR-based methods for measuring circulating miRNAs. Briefly, the procedure involves four major steps: (1 sample collection and preparation; (2 global miRNAs profiling using quantitative real-time PCR (qRT-PCR; (3 data normalization and analysis; and (4 selection and validation of miRNA biomarkers. In conclusion, qRT-PCR is a promising method for profiling of circulating miRNAs as biomarkers.

Multiplex PCR Study of Plasmid-Mediated AmpC Beta-Lactamases Genes in Clinical Isolates of Escherichia coli

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Maryam Dehghani

2017-02-01

Full Text Available Background:   AmpC β-lactamases are important cephalosporinases chromosomally encoded in many of Enterobacteriaceae and a few other organisms where they mediate resistance to cephalothin, cefazolin, cefoxitin and penicillins. The six different families of plasmid-mediated AmpC β-lactamases have been described, but no phenotypic test can discriminate among them. AmpC multiplex PCR has been successfully used to discriminate plasmid-mediated ampC specific families in organisms such as Klebsiella pneumonia and Escherichia coli. The aim of this study was to indicate the prevalence of AmpC β-lactamase genes by specifically designed primers through PCR test.Methods:   243 total clinical urine samples were collected, and 227 isolates were identified as Escherichia coli based on standard biochemical tests. Subsequently, the isolates were screened by disc diffusion and combined disc test for β-lactamase production. Resistant isolates were evaluated by PCR for ampC family determination. Results:  Antibiotic resistance pattern were observed as follows: cefepime (%25, ceftazidime (%31, ceftriaxone (%37, cefotaxime (%38. The ratio of isolates was detected as ESBLs and AmpC producers were 34% and 5.2%, respectively. PCR performed on 12 selected isolates via phenotypic tests and the results revealed that among 12 isolates, 11 contained blaCMY-42. Conclusion:  Unfortunately, antibiotic resistance has become an increasingly critical problem in many countries like Iran and occurrence of isolates co-expressing AmpC-β-lactamases and ESBLs can create serious problems in the future. As antibiotic options in the treatment of AmpC β-lactamases and ESBLs producing organisms are extremely limited, molecular screening by laboratories is suggested to reduce the risk of therapeutic defeat.

Elasmobranch qPCR reference genes: a case study of hypoxia preconditioned epaulette sharks

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Ashton Kevin J

2010-04-01

Full Text Available Abstract Background Elasmobranch fishes are an ancient group of vertebrates which have high potential as model species for research into evolutionary physiology and genomics. However, no comparative studies have established suitable reference genes for quantitative PCR (qPCR in elasmobranchs for any physiological conditions. Oxygen availability has been a major force shaping the physiological evolution of vertebrates, especially fishes. Here we examined the suitability of 9 reference candidates from various functional categories after a single hypoxic insult or after hypoxia preconditioning in epaulette shark (Hemiscyllium ocellatum. Results Epaulette sharks were caught and exposed to hypoxia. Tissues were collected from 10 controls, 10 individuals with single hypoxic insult and 10 individuals with hypoxia preconditioning (8 hypoxic insults, 12 hours apart. We produced sequence information for reference gene candidates and monitored mRNA expression levels in four tissues: cerebellum, heart, gill and eye. The stability of the genes was examined with analysis of variance, geNorm and NormFinder. The best ranking genes in our study were eukaryotic translation elongation factor 1 beta (eef1b, ubiquitin (ubq and polymerase (RNA II (DNA directed polypeptide F (polr2f. The performance of the ribosomal protein L6 (rpl6 was tissue-dependent. Notably, in one tissue the analysis of variance indicated statistically significant differences between treatments for genes that were ranked as the most stable candidates by reference gene software. Conclusions Our results indicate that eef1b and ubq are generally the most suitable reference genes for the conditions and tissues in the present epaulette shark studies. These genes could also be potential reference gene candidates for other physiological studies examining stress in elasmobranchs. The results emphasise the importance of inter-group variation in reference gene evaluation.

COMPARISON OF 16S rRNA-PCR-RFLP, LipL32-PCR AND OmpL1-PCR METHODS IN THE DIAGNOSIS OF LEPTOSPIROSIS

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Tülin GÜVEN GÖKMEN

Full Text Available SUMMARY Leptospirosis is still one of the most important health problems in developing countries located in humid tropical and subtropical regions. Human infections are generally caused by exposure to water, soil or food contaminated with the urine of infected wild and domestic animals such as rodents and dogs. The clinical course of leptospirosis is variable and may be difficult to distinguish from many other infectious diseases. The dark-field microscopy (DFM, serology and nucleic acid amplification techniques are used to diagnose leptospirosis, however, a distinctive standard reference method is still lacking. Therefore, in this study, we aimed to determine the presence of Leptospira spp., to differentiate the pathogenic L. interrogans and the non-pathogenic L. biflexa, and also to determine the sensitivity and specificity values of molecular methods as an alternative to conventional ones. A total of 133 serum samples, from 47 humans and 86 cattle were evaluated by two conventional tests: the Microagglutination Test (MAT and the DFM, as well as three molecular methods, the 16S rRNA-PCR followed by Restriction Fragment Lenght Polymorphism (RFLP of the amplification products 16S rRNA-PCR-RFLP, LipL32-PCR and OmpL1-PCR. In this study, for L. interrogans, the specificity and sensitivity rates of the 16S rRNA-PCR and the LipL32-PCR were considered similar (100% versus 98.25% and 100% versus 98.68%, respectively. The OmpL1-PCR was able to classify L. interrogans into two intergroups, but this PCR was less sensitive (87.01% than the other two PCR methods. The 16S rRNA-PCR-RFLP could detect L. biflexa DNA, but LipL32-PCR and OmpL1-PCR could not. The 16S rRNA-PCR-RFLP provided an early and accurate diagnosis and was able to distinguish pathogenic and non-pathogenic Leptospira species, hence it may be used as an alternative method to the conventional gold standard techniques for the rapid disgnosis of leptospirosis.

ICG: a wiki-driven knowledgebase of internal control genes for RT-qPCR normalization.

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Sang, Jian; Wang, Zhennan; Li, Man; Cao, Jiabao; Niu, Guangyi; Xia, Lin; Zou, Dong; Wang, Fan; Xu, Xingjian; Han, Xiaojiao; Fan, Jinqi; Yang, Ye; Zuo, Wanzhu; Zhang, Yang; Zhao, Wenming; Bao, Yiming; Xiao, Jingfa; Hu, Songnian; Hao, Lili; Zhang, Zhang

2018-01-04

Real-time quantitative PCR (RT-qPCR) has become a widely used method for accurate expression profiling of targeted mRNA and ncRNA. Selection of appropriate internal control genes for RT-qPCR normalization is an elementary prerequisite for reliable expression measurement. Here, we present ICG (http://icg.big.ac.cn), a wiki-driven knowledgebase for community curation of experimentally validated internal control genes as well as their associated experimental conditions. Unlike extant related databases that focus on qPCR primers in model organisms (mainly human and mouse), ICG features harnessing collective intelligence in community integration of internal control genes for a variety of species. Specifically, it integrates a comprehensive collection of more than 750 internal control genes for 73 animals, 115 plants, 12 fungi and 9 bacteria, and incorporates detailed information on recommended application scenarios corresponding to specific experimental conditions, which, collectively, are of great help for researchers to adopt appropriate internal control genes for their own experiments. Taken together, ICG serves as a publicly editable and open-content encyclopaedia of internal control genes and accordingly bears broad utility for reliable RT-qPCR normalization and gene expression characterization in both model and non-model organisms. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Comparison between digital PCR and real-time PCR in detection of Salmonella typhimurium in milk.

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Wang, Meng; Yang, Junjie; Gai, Zhongtao; Huo, Shengnan; Zhu, Jianhua; Li, Jun; Wang, Ranran; Xing, Sheng; Shi, Guosheng; Shi, Feng; Zhang, Lei

2018-02-02

As a kind of zero-tolerance foodborne pathogens, Salmonella typhimurium poses a great threat to quality of food products and public health. Hence, rapid and efficient approaches to identify Salmonella typhimurium are urgently needed. Combined with PCR and fluorescence technique, real-time PCR (qPCR) and digital PCR (ddPCR) are regarded as suitable tools for detecting foodborne pathogens. To compare the effect between qPCR and ddPCR in detecting Salmonella typhimurium, a series of nucleic acid, pure strain culture and spiking milk samples were applied and the resistance to inhibitors referred in this article as well. Compared with qPCR, ddPCR exhibited more sensitive (10 -4 ng/μl or 10 2 cfu/ml) and less pre-culturing time (saving 2h). Moreover, ddPCR had stronger resistance to inhibitors than qPCR, yet absolute quantification hardly performed when target's concentration over 1ng/μl or 10 6 cfu/ml. This study provides an alternative strategy in detecting foodborne Salmonella typhimurium. Copyright © 2018 Elsevier B.V. All rights reserved.

Screening for the Most Suitable Reference Genes for Gene Expression Studies in Equine Milk Somatic Cells.

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Jakub Cieslak

Full Text Available Apart from the well-known role of somatic cell count as a parameter reflecting the inflammatory status of the mammary gland, the composition of cells isolated from milk is considered as a valuable material for gene expression studies in mammals. Due to its unique composition, in recent years an increasing interest in mare's milk consumption has been observed. Thus, investigating the genetic background of horse's milk variability presents and interesting study model. Relying on 39 milk samples collected from mares representing three breeds (Polish Primitive Horse, Polish Cold-blooded Horse, Polish Warmblood Horse we aimed to investigate the utility of equine milk somatic cells as a source of mRNA and to screen the best reference genes for RT-qPCR using geNorm and NormFinder algorithms. The results showed that despite relatively low somatic cell counts in mare's milk, the amount and the quality of the extracted RNA are sufficient for gene expression studies. The analysis of the utility of 7 potential reference genes for RT-qPCR experiments for the normalization of equine milk somatic cells revealed some differences between the outcomes of the applied algorithms, although in both cases the KRT8 and TOP2B genes were pointed as the most stable. Analysis by geNorm showed that the combination of 4 reference genes (ACTB, GAPDH, TOP2B and KRT8 is required for apropriate RT-qPCR experiments normalization, whereas NormFinder algorithm pointed the combination of KRT8 and RPS9 genes as the most suitable. The trial study of the relative transcript abundance of the beta-casein gene with the use of various types and numbers of internal control genes confirmed once again that the selection of proper reference gene combinations is crucial for the final results of each real-time PCR experiment.

Pathway and network-based analysis of genome-wide association studies and RT-PCR validation in polycystic ovary syndrome.

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Shen, Haoran; Liang, Zhou; Zheng, Saihua; Li, Xuelian

2017-11-01

The purpose of this study was to identify promising candidate genes and pathways in polycystic ovary syndrome (PCOS). Microarray dataset GSE345269 obtained from the Gene Expression Omnibus database includes 7 granulosa cell samples from PCOS patients, and 3 normal granulosa cell samples. Differentially expressed genes (DEGs) were screened between PCOS and normal samples. Pathway enrichment analysis was conducted for DEGs using ClueGO and CluePedia plugin of Cytoscape. A Reactome functional interaction (FI) network of the DEGs was built using ReactomeFIViz, and then network modules were extracted, followed by pathway enrichment analysis for the modules. Expression of DEGs in granulosa cell samples was measured using quantitative RT-PCR. A total of 674 DEGs were retained, which were significantly enriched with inflammation and immune-related pathways. Eight modules were extracted from the Reactome FI network. Pathway enrichment analysis revealed significant pathways of each module: module 0, Regulation of RhoA activity and Signaling by Rho GTPases pathways shared ARHGAP4 and ARHGAP9; module 2, GlycoProtein VI-mediated activation cascade pathway was enriched with RHOG; module 3, Thromboxane A2 receptor signaling, Chemokine signaling pathway, CXCR4-mediated signaling events pathways were enriched with LYN, the hub gene of module 3. Results of RT-PCR confirmed the finding of the bioinformatic analysis that ARHGAP4, ARHGAP9, RHOG and LYN were significantly upregulated in PCOS. RhoA-related pathways, GlycoProtein VI-mediated activation cascade pathway, ARHGAP4, ARHGAP9, RHOG and LYN may be involved in the pathogenesis of PCOS.

Interlaboratory study of DNA extraction from multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for individual kernel detection system of genetically modified maize.

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Akiyama, Hiroshi; Sakata, Kozue; Makiyma, Daiki; Nakamura, Kosuke; Teshima, Reiko; Nakashima, Akie; Ogawa, Asako; Yamagishi, Toru; Futo, Satoshi; Oguchi, Taichi; Mano, Junichi; Kitta, Kazumi

2011-01-01

In many countries, the labeling of grains, feed, and foodstuff is mandatory if the genetically modified (GM) organism content exceeds a certain level of approved GM varieties. We previously developed an individual kernel detection system consisting of grinding individual kernels, DNA extraction from the individually ground kernels, GM detection using multiplex real-time PCR, and GM event detection using multiplex qualitative PCR to analyze the precise commingling level and varieties of GM maize in real sample grains. We performed the interlaboratory study of the DNA extraction with multiple ground samples, multiplex real-time PCR detection, and multiplex qualitative PCR detection to evaluate its applicability, practicality, and ruggedness for the individual kernel detection system of GM maize. DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR were evaluated by five laboratories in Japan, and all results from these laboratories were consistent with the expected results in terms of the commingling level and event analysis. Thus, the DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for the individual kernel detection system is applicable and practicable in a laboratory to regulate the commingling level of GM maize grain for GM samples, including stacked GM maize.

Evaluation of endogenous control gene(s) for gene expression studies in human blood exposed to 60Co γ-rays ex vivo

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Vaiphei, S. Thangminlal; Keppen, Joshua; Nongrum, Saibadaiahun; Sharan, R.N.; Chaubey, R.C.; Kma, L.

2015-01-01

In gene expression studies, it is critical to normalize data using a stably expressed endogenous control gene in order to obtain accurate and reliable results. However, we currently do not have a universally applied endogenous control gene for normalization of data for gene expression studies, particularly those involving 60 Co γ-ray-exposed human blood samples. In this study, a comparative assessment of the gene expression of six widely used housekeeping endogenous control genes, namely 18S, ACTB, B2M, GAPDH, MT-ATP6 and CDKN1A, was undertaken for a range of 60 Co γ-ray doses (0.5, 1.0, 2.0 and 4.0 Gy) at 8.4 Gy min -1 at 0 and 24 h post-irradiation time intervals. Using the NormFinder algorithm, real-time PCR data obtained from six individuals (three males and three females) were analyzed with respect to the threshold cycle (Ct) value and abundance, ΔCt pair-wise comparison, intra- and inter-group variability assessments, etc. GAPDH, either alone or in combination with 18S, was found to be the most suitable endogenous control gene and should be used in gene expression studies, especially those involving qPCR of γ-ray-exposed human blood samples. (author)

Comparative evaluation of conventional RT-PCR and real-time RT-PCR (RRT-PCR) for detection of avian metapneumovirus subtype A

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Ferreira, HL; Spilki, FR; dos Santos, MMAB; de Almeida, RS; Arns, CW

2009-01-01

Avian metapneumovirus (AMPV) belongs to Metapneumovirus genus of Paramyxoviridae family. Virus isolation, serology, and detection of genomic RNA are used as diagnostic methods for AMPV. The aim of the present study was to compare the detection of six subgroup A AMPV isolates (AMPV/A) viral RNA by using different conventional and real time RT-PCR methods. Two new RT-PCR tests and two real time RT-PCR tests, both detecting fusion (F) gene and nucleocapsid (N) gene were compared with an establis...

Cloning and expression of NS3 helicase fragment of hepatitis C virus and the study of its immunoreactivity in HCV infected patients

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Mahrou Sadri

2015-02-01

Full Text Available Objective(s: Hepatitis C is a major cause of liver failure worldwide. Current therapies applied for this disease are not fully effective and produce side effects in most cases. Non-structural protein 3 helicase (NS3 of HCV is one of the key enzymes in viral replication and infection. Therefore, this region is a promising target to design new drugs and therapies against HCV infection. The aim of this study was cloning and expression of HCV NS3 helicase fragment in Escherichia coli BL21 (DE3 using pET102/D-TOPO expression vector and studying immunoreactivity of the expressed antigen in Iranian infected with hepatitis C. Materials and Methods: The viral RNA was extracted from the serum of HCV infected patient. The NS3 helicase region was amplified by RT-PCR. The PCR product was directionally cloned into the expression vector pET102/D-TOPO and transformed into the BL21 strain of E. coli (DE3. The transformed bacteria were then induced by adding 1mM isopropyl-β-D-thiogalactopyranoside (IPTG into the culture medium to enhance the protein expression. SDS-PAGE and western blotting were carried out to identify the protein under investigation, and finally purified recombinant fusion protein was used as the antigen for ELISA method. Results: Theinsertion of theDNA fragment of the NS3 regioninto the expression vectorwas further confirmed by PCR and sequencing. SDS-PAGE analysis showed the successful expression of the recombinant protein of interest. Furthermore, immunoreactivity of fusion NS3 helicase was confirmed by ELISA and western blotting. Conclusion: It seems that this recombinant protein could be a useful source of antigen for future studies on HCV diagnosis and therapy.

Analytical Performance of Four Polymerase Chain Reaction (PCR and Real Time PCR (qPCR Assays for the Detection of Six Leishmania Species DNA in Colombia

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Cielo M. León

2017-10-01

Full Text Available Leishmaniasis comprises a spectrum of parasitic diseases caused by protozoans of the genus Leishmania. Molecular tools have been widely employed for the detection of Leishmania due to its high sensitivity and specificity. However, the analytical performance of molecular platforms as PCR and real time PCR (qPCR including a wide variety of molecular markers has never been evaluated. Herein, the aim was to evaluate the analytical performance of 4 PCR-based assays (designed on four different targets and applied on conventional and real-time PCR platforms. We evaluated the analytical performance of conventional PCR and real time PCR, determining exclusivity and inclusivity, Anticipated Reportable Range (ARR, limit of detection (LoD and accuracy using primers directed to kDNA, HSP70, 18S and ITS-1 targets. We observed that the kDNA was the most sensitive but does not meet the criterion of exclusivity. The HSP70 presented a higher LoD in conventional PCR and qPCR in comparison with the other markers (1 × 101 and 1 × 10-1 equivalent parasites/mL respectively and had a higher coefficient of variation in qPCR. No statistically significant differences were found between the days of the test with the four molecular markers. The present study revealed that the 18S marker presented the best performance in terms of analytical sensitivity and specificity for the qPCR in the species tested (species circulating in Colombia. Therefore, we recommend to explore the analytical and diagnostic performance in future studies using a broader number of species across America.

Analytical Performance of Four Polymerase Chain Reaction (PCR) and Real Time PCR (qPCR) Assays for the Detection of Six Leishmania Species DNA in Colombia

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León, Cielo M.; Muñoz, Marina; Hernández, Carolina; Ayala, Martha S.; Flórez, Carolina; Teherán, Aníbal; Cubides, Juan R.; Ramírez, Juan D.

2017-01-01

Leishmaniasis comprises a spectrum of parasitic diseases caused by protozoans of the genus Leishmania. Molecular tools have been widely employed for the detection of Leishmania due to its high sensitivity and specificity. However, the analytical performance of molecular platforms as PCR and real time PCR (qPCR) including a wide variety of molecular markers has never been evaluated. Herein, the aim was to evaluate the analytical performance of 4 PCR-based assays (designed on four different targets) and applied on conventional and real-time PCR platforms. We evaluated the analytical performance of conventional PCR and real time PCR, determining exclusivity and inclusivity, Anticipated Reportable Range (ARR), limit of detection (LoD) and accuracy using primers directed to kDNA, HSP70, 18S and ITS-1 targets. We observed that the kDNA was the most sensitive but does not meet the criterion of exclusivity. The HSP70 presented a higher LoD in conventional PCR and qPCR in comparison with the other markers (1 × 101 and 1 × 10-1 equivalent parasites/mL respectively) and had a higher coefficient of variation in qPCR. No statistically significant differences were found between the days of the test with the four molecular markers. The present study revealed that the 18S marker presented the best performance in terms of analytical sensitivity and specificity for the qPCR in the species tested (species circulating in Colombia). Therefore, we recommend to explore the analytical and diagnostic performance in future studies using a broader number of species across America. PMID:29046670

Reference gene selection for real-time quantitative PCR analysis of the mouse uterus in the peri-implantation period.

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Pengfei Lin

Full Text Available The study of uterine gene expression patterns is valuable for understanding the biological and molecular mechanisms that occur during embryo implantation. Real-time quantitative RT-PCR (qRT-PCR is an extremely sensitive technique that allows for the precise quantification of mRNA abundance; however, selecting stable reference genes suitable for the normalization of qRT-PCR data is required to avoid the misinterpretation of experimental results and erroneous analyses. This study employs several mouse models, including an early pregnancy, a pseudopregnancy, a delayed implantation and activation, an artificial decidualization and a hormonal treatment model; ten candidate reference genes (PPIA, RPLP0, HPRT1, GAPDH, ACTB, TBP, B2M, 18S, UBC and TUBA that are found in uterine tissues were assessed for their suitability as internal controls for relative qRT-PCR quantification. GeNorm(PLUS, NormFinder, and BestKeeper were used to evaluate these candidate reference genes, and all of these methods identified RPLP0 and GAPDH as the most stable candidates and B2M and 18S as the least stable candidates. However, when the different models were analyzed separately, the reference genes exhibited some variation in their expression levels.

Tissue- and cell-specific expression of metallothionein genes in cadmium- and copper-exposed mussels analyzed by in situ hybridization and RT-PCR

International Nuclear Information System (INIS)

Zorita, I.; Bilbao, E.; Schad, A.; Cancio, I.; Soto, M.; Cajaraville, M.P.

2007-01-01

Metallothioneins (MTs) are metal-inducible proteins that can be used as biomarkers of metal exposure. In mussels two families of MT isoforms (MT10 and MT20) have been characterized. In this study, mussels (Mytilus galloprovincialis) were exposed to 200 ppb Cd and 40 ppb Cu for 2 and 9 days to characterize the tissue and isoform specificity of metal-induced MT expression. Non-radioactive in situ hybridization demonstrated that both MT isoforms were mainly transcribed in digestive tubule epithelial cells, especially in basophilic cells. Weaker MT expression was detected in non-ciliated duct cells, stomach and gill epithelial cells, haemocytes, adipogranular cells, spermatic follicles and oocytes. RT-PCR resulted in cloning of a novel M. galloprovincialis isoform homologous to recently cloned Mytilus edulis intron-less MT10B isoform. In gills, Cd only affected MT10 gene expression after 2 days of exposure while increases in MT protein levels occurred at day 9. In the digestive gland, a marked increase of both isoforms, but especially of MT20, was accompanied by increased levels of MT proteins and basophilic cell volume density (Vv BAS ) after 2 and 9 days and of intralysosomal metal accumulation in digestive cells after 9 days. Conversely, although metal was accumulated in digestive cells lysosomes and the Vv BAS increased in Cu-exposed mussels, Cu exposure did not produce an increase of MT gene expression or MT protein levels. These data suggest that MTs are expressed in a tissue-, cell- and isoform-specific way in response to different metals

Identification and evaluation of new reference genes in Gossypium hirsutum for accurate normalization of real-time quantitative RT-PCR data

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Alves-Ferreira Marcio

2010-03-01

Full Text Available Abstract Background Normalizing through reference genes, or housekeeping genes, can make more accurate and reliable results from reverse transcription real-time quantitative polymerase chain reaction (qPCR. Recent studies have shown that no single housekeeping gene is universal for all experiments. Thus, suitable reference genes should be the first step of any qPCR analysis. Only a few studies on the identification of housekeeping gene have been carried on plants. Therefore qPCR studies on important crops such as cotton has been hampered by the lack of suitable reference genes. Results By the use of two distinct algorithms, implemented by geNorm and NormFinder, we have assessed the gene expression of nine candidate reference genes in cotton: GhACT4, GhEF1α5, GhFBX6, GhPP2A1, GhMZA, GhPTB, GhGAPC2, GhβTUB3 and GhUBQ14. The candidate reference genes were evaluated in 23 experimental samples consisting of six distinct plant organs, eight stages of flower development, four stages of fruit development and in flower verticils. The expression of GhPP2A1 and GhUBQ14 genes were the most stable across all samples and also when distinct plants organs are examined. GhACT4 and GhUBQ14 present more stable expression during flower development, GhACT4 and GhFBX6 in the floral verticils and GhMZA and GhPTB during fruit development. Our analysis provided the most suitable combination of reference genes for each experimental set tested as internal control for reliable qPCR data normalization. In addition, to illustrate the use of cotton reference genes we checked the expression of two cotton MADS-box genes in distinct plant and floral organs and also during flower development. Conclusion We have tested the expression stabilities of nine candidate genes in a set of 23 tissue samples from cotton plants divided into five different experimental sets. As a result of this evaluation, we recommend the use of GhUBQ14 and GhPP2A1 housekeeping genes as superior references

Identification and evaluation of new reference genes in Gossypium hirsutum for accurate normalization of real-time quantitative RT-PCR data.

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Artico, Sinara; Nardeli, Sarah M; Brilhante, Osmundo; Grossi-de-Sa, Maria Fátima; Alves-Ferreira, Marcio

2010-03-21

Normalizing through reference genes, or housekeeping genes, can make more accurate and reliable results from reverse transcription real-time quantitative polymerase chain reaction (qPCR). Recent studies have shown that no single housekeeping gene is universal for all experiments. Thus, suitable reference genes should be the first step of any qPCR analysis. Only a few studies on the identification of housekeeping gene have been carried on plants. Therefore qPCR studies on important crops such as cotton has been hampered by the lack of suitable reference genes. By the use of two distinct algorithms, implemented by geNorm and NormFinder, we have assessed the gene expression of nine candidate reference genes in cotton: GhACT4, GhEF1alpha5, GhFBX6, GhPP2A1, GhMZA, GhPTB, GhGAPC2, GhbetaTUB3 and GhUBQ14. The candidate reference genes were evaluated in 23 experimental samples consisting of six distinct plant organs, eight stages of flower development, four stages of fruit development and in flower verticils. The expression of GhPP2A1 and GhUBQ14 genes were the most stable across all samples and also when distinct plants organs are examined. GhACT4 and GhUBQ14 present more stable expression during flower development, GhACT4 and GhFBX6 in the floral verticils and GhMZA and GhPTB during fruit development. Our analysis provided the most suitable combination of reference genes for each experimental set tested as internal control for reliable qPCR data normalization. In addition, to illustrate the use of cotton reference genes we checked the expression of two cotton MADS-box genes in distinct plant and floral organs and also during flower development. We have tested the expression stabilities of nine candidate genes in a set of 23 tissue samples from cotton plants divided into five different experimental sets. As a result of this evaluation, we recommend the use of GhUBQ14 and GhPP2A1 housekeeping genes as superior references for normalization of gene expression measures in

Evaluation of methods for oligonucleotide array data via quantitative real-time PCR.

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Qin, Li-Xuan; Beyer, Richard P; Hudson, Francesca N; Linford, Nancy J; Morris, Daryl E; Kerr, Kathleen F

2006-01-17

There are currently many different methods for processing and summarizing probe-level data from Affymetrix oligonucleotide arrays. It is of great interest to validate these methods and identify those that are most effective. There is no single best way to do this validation, and a variety of approaches is needed. Moreover, gene expression data are collected to answer a variety of scientific questions, and the same method may not be best for all questions. Only a handful of validation studies have been done so far, most of which rely on spike-in datasets and focus on the question of detecting differential expression. Here we seek methods that excel at estimating relative expression. We evaluate methods by identifying those that give the strongest linear association between expression measurements by array and the "gold-standard" assay. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is generally considered the "gold-standard" assay for measuring gene expression by biologists and is often used to confirm findings from microarray data. Here we use qRT-PCR measurements to validate methods for the components of processing oligo array data: background adjustment, normalization, mismatch adjustment, and probeset summary. An advantage of our approach over spike-in studies is that methods are validated on a real dataset that was collected to address a scientific question. We initially identify three of six popular methods that consistently produced the best agreement between oligo array and RT-PCR data for medium- and high-intensity genes. The three methods are generally known as MAS5, gcRMA, and the dChip mismatch mode. For medium- and high-intensity genes, we identified use of data from mismatch probes (as in MAS5 and dChip mismatch) and a sequence-based method of background adjustment (as in gcRMA) as the most important factors in methods' performances. However, we found poor reliability for methods using mismatch probes for low-intensity genes

Evaluation of methods for oligonucleotide array data via quantitative real-time PCR

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Morris Daryl E

2006-01-01

Full Text Available Abstract Background There are currently many different methods for processing and summarizing probe-level data from Affymetrix oligonucleotide arrays. It is of great interest to validate these methods and identify those that are most effective. There is no single best way to do this validation, and a variety of approaches is needed. Moreover, gene expression data are collected to answer a variety of scientific questions, and the same method may not be best for all questions. Only a handful of validation studies have been done so far, most of which rely on spike-in datasets and focus on the question of detecting differential expression. Here we seek methods that excel at estimating relative expression. We evaluate methods by identifying those that give the strongest linear association between expression measurements by array and the "gold-standard" assay. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR is generally considered the "gold-standard" assay for measuring gene expression by biologists and is often used to confirm findings from microarray data. Here we use qRT-PCR measurements to validate methods for the components of processing oligo array data: background adjustment, normalization, mismatch adjustment, and probeset summary. An advantage of our approach over spike-in studies is that methods are validated on a real dataset that was collected to address a scientific question. Results We initially identify three of six popular methods that consistently produced the best agreement between oligo array and RT-PCR data for medium- and high-intensity genes. The three methods are generally known as MAS5, gcRMA, and the dChip mismatch mode. For medium- and high-intensity genes, we identified use of data from mismatch probes (as in MAS5 and dChip mismatch and a sequence-based method of background adjustment (as in gcRMA as the most important factors in methods' performances. However, we found poor reliability for methods

Performance of a RT-PCR Assay in Comparison to FISH and Immunohistochemistry for the Detection of ALK in Non-Small Cell Lung Cancer.

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Hout, David R; Schweitzer, Brock L; Lawrence, Kasey; Morris, Stephan W; Tucker, Tracy; Mazzola, Rosetta; Skelton, Rachel; McMahon, Frank; Handshoe, John; Lesperance, Mary; Karsan, Aly; Saltman, David L

2017-08-01

Patients with lung cancers harboring an activating anaplastic lymphoma kinase ( ALK ) rearrangement respond favorably to ALK inhibitor therapy. Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) are validated and widely used screening tests for ALK rearrangements but both methods have limitations. The ALK RGQ RT-PCR Kit (RT-PCR) is a single tube quantitative real-time PCR assay for high throughput and automated interpretation of ALK expression. In this study, we performed a direct comparison of formalin-fixed paraffin-embedded (FFPE) lung cancer specimens using all three ALK detection methods. The RT-PCR test (diagnostic cut-off Δ C t of ≤8) was shown to be highly sensitive (100%) when compared to FISH and IHC. Sequencing of RNA detected full-length ALK transcripts or EML4-ALK and KIF5B-ALK fusion variants in discordant cases in which ALK expression was detected by the ALK RT-PCR test but negative by FISH and IHC. The overall specificity of the RT-PCR test for the detection of ALK in cases without full-length ALK expression was 94% in comparison to FISH and sequencing. These data support the ALK RT-PCR test as a highly efficient and reliable diagnostic screening approach to identify patients with non-small cell lung cancer whose tumors are driven by oncogenic ALK.

Differentiation of bacterial feeding nematodes in soil ecological studies by means of arbitrarily primed PCR

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Van Der Knaap, Esther; Rodriguez, Russell J.; Freckman, Diana W.

1993-01-01

Arbitrarily-primed polymerase chain reaction (ap-PCR) was used to differentiate closely related bacterial-feeding nematodes of the genera: Caenorhabditis, Acrobeloides, Cephalobus and Zeldia. Average percentage similarity of bands generated by ap-PCR with seven different primers between 14 isolates of Caenorhabditis elegans was ⪢ 90%, whereas between C. elegans, C. briggsae and C. remanei similarity was nematode populations were also obtained from ap-PCR analysis of single worms. Due to the difficulty of identification of soil nematodes, the ap-PCR offers potential as a rapid and reliable technique to assess biodiversity. Ap-PCR will make it feasible, for the first time, to study the ecological interactions of unique nematode genotypes in soil habitats.

Quantitative real-time PCR approaches for microbial community studies in wastewater treatment systems: applications and considerations.

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Kim, Jaai; Lim, Juntaek; Lee, Changsoo

2013-12-01

Quantitative real-time PCR (qPCR) has been widely used in recent environmental microbial ecology studies as a tool for detecting and quantifying microorganisms of interest, which aids in better understandings of the complexity of wastewater microbial communities. Although qPCR can be used to provide more specific and accurate quantification than other molecular techniques, it does have limitations that must be considered when applying it in practice. This article reviews the principle of qPCR quantification and its applications to microbial ecology studies in various wastewater treatment environments. Here we also address several limitations of qPCR-based approaches that can affect the validity of quantification data: template nucleic acid quality, nucleic acid extraction efficiency, specificity of group-specific primers and probes, amplification of nonviable DNA, gene copy number variation, and limited number of sequences in the database. Even with such limitations, qPCR is reportedly among the best methods for quantitatively investigating environmental microbial communities. The application of qPCR is and will continue to be increasingly common in studies of wastewater treatment systems. To obtain reliable analyses, however, the limitations that have often been overlooked must be carefully considered when interpreting the results. Copyright © 2013 Elsevier Inc. All rights reserved.

Expression of the mucus adhesion genes Mub and MapA, adhesion-like factor EF-Tu and bacteriocin gene plaA of Lactobacillus plantarum 423, monitored with real-time PCR.

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Ramiah, K; van Reenen, C A; Dicks, L M T

2007-05-30

Expression of the mucus adhesion genes Mub and MapA, adhesion-like factor EF-Tu and bacteriocin gene plaA by Lactobacillus plantarum 423, grown in the presence of bile, pancreatin and at low pH, was studied by real-time PCR. Mub, MapA and EF-Tu were up-regulated in the presence of mucus, proportional to increasing concentrations. Expression of MapA was up-regulated in the presence of 3.0 g/l bile and 3.0 g/l pancreatin at pH 6.5. Similar results were recorded in the presence of 10.0 g/l bile and 10.0 g/l pancreatin at pH 6.5. Expression of Mub was down-regulated in the presence of bile and pancreatin, whilst the expression of EF-Tu and plaA remained unchanged. Expression of Mub and MapA remained unchanged at pH 4.0, whilst expression of EF-Tu and plaA were up-regulated. Expression of MapA was down-regulated in the presence of 1.0 g/l l-cysteine HCl, suggesting that the gene is regulated by transcription attenuation that involves cysteine.

Selection of Suitable Reference Genes for Quantitative Real-time PCR in Sapium sebiferum

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Xue Chen

2017-05-01

Full Text Available Chinese tallow (Sapium sebiferum L. is a promising landscape and bioenergy plant. Measuring gene expression by quantitative real-time polymerase chain reaction (qRT-PCR can provide valuable information on gene function. Stably expressed reference genes for normalization are a prerequisite for ensuring the accuracy of the target gene expression level among different samples. However, the reference genes in Chinese tallow have not been systematically validated. In this study, 12 candidate reference genes (18S, GAPDH, UBQ, RPS15, SAND, TIP41, 60S, ACT7, PDF2, APT, TBP, and TUB were investigated with qRT-PCR in 18 samples, including those from different tissues, from plants treated with sucrose and cold stresses. The data were calculated with four common algorithms, geNorm, BestKeeper, NormFinder, and the delta cycle threshold (ΔCt. TIP41 and GAPDH were the most stable for the tissue-specific experiment, GAPDH and 60S for cold treatment, and GAPDH and UBQ for sucrose stresses, while the least stable genes were 60S, TIP41, and 18S respectively. The comprehensive results showed APT, GAPDH, and UBQ to be the top-ranked stable genes across all the samples. The stability of 60S was the lowest during all experiments. These selected reference genes were further validated by comparing the expression profiles of the chalcone synthase gene in Chinese tallow in different samples. The results will help to improve the accuracy of gene expression studies in Chinese tallow.

Development of PCR for WWTP based on a case study

International Nuclear Information System (INIS)

Del Borghi, A.; Gallo, M.; Strazza, C.; Gaggero, P.L.

2009-01-01

In order to apply the Environmental Product Declaration (EPD) to products or services, specific rules (PCR, Product Category Rules) have to be issued to ensure comparability among different declarations within the same service group. The aim of the present study is to describe the reasons leading to each choice in the development of PCR applied to collecting and treatment service of municipal wastewater and to evaluate, through life cycle analysis (LCA), their influence on the potential environmental impact of a wastewater treatment plant (WWTP), analysed as a case study. Specific data were collected during the year 2003 from an Italian active sludge treatment plant with separate wastewater and sludge treatment lines. The life-cycle approach applied to the case study shows that LCA represents an environmental management tool able both to communicate environmental information by the Type 3. environmental label EPD, and to look for different scenarios that can improve the environmental performance of the Collecting and Treatment Service of Municipal Wastewater. [it

Identification and validation of reference genes for quantitative real-time PCR in Drosophila suzukii (Diptera: Drosophilidae.

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Yifan Zhai

Full Text Available To accurately evaluate gene expression levels and obtain more accurate quantitative real-time RT-PCR (qRT-PCR data, normalization relative to reliable reference gene(s is required. Drosophila suzukii, is an invasive fruit pest native to East Asia, and recently invaded Europe and North America, the stability of its reference genes have not been previously investigated. In this study, ten candidate reference genes (RPL18, RPS3, AK, EF-1β, TBP, NADH, HSP22, GAPDH, Actin, α-Tubulin, were evaluated for their suitability as normalization genes under different biotic (developmental stage, tissue and population, and abiotic (photoperiod, temperature conditions. The three statistical approaches (geNorm, NormFinder and BestKeeper and one web-based comprehensive tool (RefFinder were used to normalize analysis of the ten candidate reference genes identified α-Tubulin, TBP and AK as the most stable candidates, while HSP22 and Actin showed the lowest expression stability. We used three most stable genes (α-Tubulin, TBP and AK and one unstably expressed gene to analyze the expression of P-glycoprotein in abamectin-resistant and sensitive strains, and the results were similar to reference genes α-Tubulin, TBP and AK, which show good stability, while the result of HSP22 has a certain bias. The three validated reference genes can be widely used for quantification of target gene expression with qRT-PCR technology in D.suzukii.

Optimisation of the RT-PCR detection of immunomagnetically enriched carcinoma cells

International Nuclear Information System (INIS)

Raynor, Michael; Stephenson, Sally-Anne; Walsh, David CA; Pittman, Kenneth B; Dobrovic, Alexander

2002-01-01

Immunomagnetic enrichment followed by RT-PCR (immunobead RT-PCR) is an efficient methodology to identify disseminated carcinoma cells in the blood and bone marrow. The RT-PCR assays must be both specific for the tumor cells and sufficiently sensitive to enable detection of single tumor cells. We have developed a method to test RT-PCR assays for any cancer. This has been investigated using a panel of RT-PCR markers suitable for the detection of breast cancer cells. In the assay, a single cell line-derived tumor cell is added to 100 peripheral blood mononuclear cells (PBMNCs) after which mRNA is isolated and reverse transcribed for RT-PCR analysis. PBMNCs without added tumor cells are used as specificity controls. The previously studied markers epidermal growth factor receptor (EGFR), mammaglobin 1 (MGB1), epithelial cell adhesion molecule (EpCAM/TACSTD1), mucin 1 (MUC1), carcinoembryonic antigen (CEA) were tested. Two new epithelial-specific markers ELF3 and EphB4 were also tested. MUC1 was unsuitable as strong amplification was detected in 100 cell PBMNC controls. Expression of ELF3, EphB4, EpCAM, EGFR, CEA and MGB1 was found to be both specific for the tumor cell, as demonstrated by the absence of a signal in most 100 cell PBMNC controls, and sensitive enough to detect a single tumor cell in 100 PBMNCs using a single round of RT-PCR. ELF3, EphB4, EpCAM, EGFR, CEA and MGB1 are appropriate RT-PCR markers for use in a marker panel to detect disseminated breast cancer cells after immunomagnetic enrichment

High Throughput qPCR Expression Profiling of Circulating MicroRNAs Reveals Minimal Sex- and Sample Timing-Related Variation in Plasma of Healthy Volunteers.

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Catherine Mooney

Full Text Available MicroRNAs are a class of small non-coding RNA that regulate gene expression at a post-transcriptional level. MicroRNAs have been identified in various body fluids under normal conditions and their stability as well as their dysregulation in disease opens up a new field for biomarker study. However, diurnal and day-to-day variation in plasma microRNA levels, and differential regulation between males and females, may affect biomarker stability. A QuantStudio 12K Flex Real-Time PCR System was used to profile plasma microRNA levels using OpenArray in male and female healthy volunteers, in the morning and afternoon, and at four time points over a one month period. Using this system we were able to run four OpenArray plates in a single run, the equivalent of 32 traditional 384-well qPCR plates or 12,000 data points. Up to 754 microRNAs can be identified in a single plasma sample in under two hours. 108 individual microRNAs were identified in at least 80% of all our samples which compares favourably with other reports of microRNA profiles in serum or plasma in healthy adults. Many of these microRNAs, including miR-16-5p, miR-17-5p, miR-19a-3p, miR-24-3p, miR-30c-5p, miR-191-5p, miR-223-3p and miR-451a are highly expressed and consistent with previous studies using other platforms. Overall, microRNA levels were very consistent between individuals, males and females, and time points and we did not detect significant differences in levels of microRNAs. These results suggest the suitability of this platform for microRNA profiling and biomarker discovery and suggest minimal confounding influence of sex or sample timing. However, the platform has not been subjected to rigorous validation which must be demonstrated in future biomarker studies where large differences may exist between disease and control samples.

A three-gene expression signature model for risk stratification of patients with neuroblastoma.

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Garcia, Idoia; Mayol, Gemma; Ríos, José; Domenech, Gema; Cheung, Nai-Kong V; Oberthuer, André; Fischer, Matthias; Maris, John M; Brodeur, Garrett M; Hero, Barbara; Rodríguez, Eva; Suñol, Mariona; Galvan, Patricia; de Torres, Carmen; Mora, Jaume; Lavarino, Cinzia

2012-04-01

Neuroblastoma is an embryonal tumor with contrasting clinical courses. Despite elaborate stratification strategies, precise clinical risk assessment still remains a challenge. The purpose of this study was to develop a PCR-based predictor model to improve clinical risk assessment of patients with neuroblastoma. The model was developed using real-time PCR gene expression data from 96 samples and tested on separate expression data sets obtained from real-time PCR and microarray studies comprising 362 patients. On the basis of our prior study of differentially expressed genes in favorable and unfavorable neuroblastoma subgroups, we identified three genes, CHD5, PAFAH1B1, and NME1, strongly associated with patient outcome. The expression pattern of these genes was used to develop a PCR-based single-score predictor model. The model discriminated patients into two groups with significantly different clinical outcome [set 1: 5-year overall survival (OS): 0.93 ± 0.03 vs. 0.53 ± 0.06, 5-year event-free survival (EFS): 0.85 ± 0.04 vs. 0.042 ± 0.06, both P model was an independent marker for survival (P model robustly classified patients in the total cohort and in different clinically relevant risk subgroups. We propose for the first time in neuroblastoma, a technically simple PCR-based predictor model that could help refine current risk stratification systems. ©2012 AACR.

Quantitative real-time PCR analysis of Anopheles dirus TEP1 and NOS during Plasmodium berghei infection, using three reference genes

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Jonathan W.K. Liew

2017-07-01

Full Text Available Quantitative reverse transcription PCR (qRT-PCR has been an integral part of characterizing the immunity of Anopheles mosquitoes towards Plasmodium invasion. Two anti-Plasmodium factors of Anopheles, thioester-containing protein 1 (TEP1 and nitric oxide synthase (NOS, play a role in the refractoriness of Anopheles towards Plasmodium infection and are generally expressed during infection. However, these are less studied in Anopheles dirus, a dominant malaria vector in Southeast Asia. Furthermore, most studies used a single reference gene for normalization during gene expression analysis without proper validation. This may lead to erroneous quantification of expression levels. Therefore, the present study characterized and investigated the expression profiles of TEP1 and NOS of Anopheles dirus during P. berghei infection. Prior to that, the elongation factor 1-alpha (EF1, actin 1 (Act and ribosomal protein S7 (S7 genes were validated for their suitability as a set of reference genes. TEP1 and NOS expressions in An. dirus were found to be significantly induced after P. berghei infection.

A comparative study of digital PCR and real-time qPCR for the detection and quantification of HPV mRNA in sentinel lymph nodes of cervical cancer patients.

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Carow, Katrin; Read, Christina; Häfner, Norman; Runnebaum, Ingo B; Corner, Adam; Dürst, Matthias

2017-10-30

Qualitative analyses showed that the presence of HPV mRNA in sentinel lymph nodes of cervical cancer patients with pN0 status is associated with significantly decreased recurrence free survival. To further address the clinical potential of the strategy and to define prognostic threshold levels it is necessary to use a quantitative assay. Here, we compare two methods of quantification: digital PCR and standard quantitative PCR. Serial dilutions of 5 ng-5 pg RNA (≙ 500-0.5 cells) of the cervical cancer cell line SiHa were prepared in 5 µg RNA of the HPV-negative human keratinocyte cell line HaCaT. Clinical samples consisted of 10 sentinel lymph nodes with varying HPV transcript levels. Reverse transcription of total RNA (5 µg RNA each) was performed in 100 µl and cDNA aliquots were analyzed by qPCR and dPCR. Digital PCR was run in the RainDrop ® Digital PCR system (RainDance Technologies) using a probe-based detection of HPV E6/E7 cDNA PCR products with 11 µl template. qPCR was done using a Rotor Gene Q 5plex HRM (Qiagen) amplifying HPV E6/E7 cDNA in a SYBR Green format with 1 µl template. For the analysis of both, clinical samples and serial dilution samples, dPCR and qPCR showed comparable sensitivity. With regard to reproducibility, both methods differed considerably, especially for low template samples. Here, we found with qPCR a mean variation coefficient of 126% whereas dPCR enabled a significantly lower mean variation coefficient of 40% (p = 0.01). Generally, we saw with dPCR a substantial reduction of subsampling errors, which most likely reflects the large cDNA amounts available for analysis. Compared to real-time PCR, dPCR shows higher reliability. Thus, our HPV mRNA dPCR assay holds promise for the clinical evaluation of occult tumor cells in histologically tumor-free lymph nodes in future studies.

Identification and validation of reference genes for quantitative RT-PCR normalization in wheat

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Porceddu Enrico

2009-02-01

Full Text Available Abstract Background Usually the reference genes used in gene expression analysis have been chosen for their known or suspected housekeeping roles, however the variation observed in most of them hinders their effective use. The assessed lack of validated reference genes emphasizes the importance of a systematic study for their identification. For selecting candidate reference genes we have developed a simple in silico method based on the data publicly available in the wheat databases Unigene and TIGR. Results The expression stability of 32 genes was assessed by qRT-PCR using a set of cDNAs from 24 different plant samples, which included different tissues, developmental stages and temperature stresses. The selected sequences included 12 well-known HKGs representing different functional classes and 20 genes novel with reference to the normalization issue. The expression stability of the 32 candidate genes was tested by the computer programs geNorm and NormFinder using five different data-sets. Some discrepancies were detected in the ranking of the candidate reference genes, but there was substantial agreement between the groups of genes with the most and least stable expression. Three new identified reference genes appear more effective than the well-known and frequently used HKGs to normalize gene expression in wheat. Finally, the expression study of a gene encoding a PDI-like protein showed that its correct evaluation relies on the adoption of suitable normalization genes and can be negatively affected by the use of traditional HKGs with unstable expression, such as actin and α-tubulin. Conclusion The present research represents the first wide screening aimed to the identification of reference genes and of the corresponding primer pairs specifically designed for gene expression studies in wheat, in particular for qRT-PCR analyses. Several of the new identified reference genes outperformed the traditional HKGs in terms of expression stability

Studies of Wilms’ Tumor (WT1 Gene Expression in Adult Acute Leukemias in Singapore

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Che Kang Lim

2007-01-01

Full Text Available Biomarkers provide certain values for diagnosis, monitor treatment effi cacy, or for the development of novel therapeutic approach for particular diseases. Thus, the identifi cation of specifi c of biomarkers for specifi c medical problems, including malignant diseases may be valuable in medical practice. In the study, we have used the Wilms’ tumor gene (WT1 as a biomarker to evaluate its expression in local adult patients with newly diagnosed acute leukemia, including both acute myeloid and lymphoid leukemias (AML and ALL.Aim: To investigate WT1 gene expression in adult patients with acute leukemia at diagnosis.Methods: Eighteen patients with acute leukemia diagnosed at Singapore General Hospital, Singapore, between September, 2004 and July, 2005 were included in this study. There were fifteen AML and three ALL cases aged from 18 to 71 years old. Total RNA and DNA was extracted from peripheral blood mononuclear cells (PBMCs. Expression of WT1 was detected by nested reverse-transcription polymerase chain reaction (Nested RT-PCR. K562, and 3T3 cells were used as positive- and negative-controls. The results were revalidated using real-time PCR. HLA-A genotyping was performed using sequence specific oligonucleotide polymorphism (SSOP analysis.Results: WT1 gene was exclusively expressed in all eighteen, including three ALL and fi fteen AML, patients. In contrast with WT1 gene, the HLA-A genotyping was remarkably heterogeneous in these patients.Conclusions: WT1 gene expression was observed in local patients with acute leukemia at diagnosis. It may be used as a potential molecular marker for diagnosis, clinical progression of the diseases or monitoring the response to treatment, as well as a target for the development of novel therapeutic approaches.

Human Interleukine-1 receptor antagonist:Cloning, Expression and Optimization in E.coli Host

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Gh. Barati

2014-07-01

Full Text Available Introduction & Objective: Interleukine-1 receptor antagonist (IL-1RA is a powerful anti-inflammatory cytokine which limits the biological effects of IL-1. Due to structural similarity between IL-1 and its antagonist, IL-1RA competitively binds to IL-1 receptor which leads to no signal transduction. Therefore , it is applied in the treatment of patients with inflammatory diseases such as Rheumatoid Arthritis. The aim of this study is cloning, expression and op-timization of IL-1RA in E. coli. Materials & Methods: In this experimental study synthetically prepared cDNA was amplified by PCR. After double digestion with NdeI and XhoI restriction enzymes, this gene was cloned in pET28a expression vector. Expression of desired gene was analyzed at RNA level by RT-PCR and at protein level by SDS-PAGE and followed by western blot to confirm SDS-PAGE results. Optimization of recombinant protein expression was performed in dif-ferent IPTG concentrations and harvesting times after induction. Results: The presence of gene in pET28a was determined by colony-PCR and confirmed by restriction digestion. Transcription of cloned gene and expression of high yield recombinant protein were shown by RT-PCR and SDS-PAGE, respectively. The result of SDS-PAGE was confirmed by western blot. Expression was optimized in different induction time and IPTG concentrations Conclusion: The result of this study demonstrated expression of this recombinant protein at high level in E.coli system by pET28a expression vector. This study also showed a direct as-sociation between the increased level of expression and time of induction . Therefore, an overnight induction time with 0.1 mM IPTG concentration is recommended for a high level expression. (Sci J Hamadan Univ Med Sci 2014; 21 (2:145-151

Selection of internal control genes for real-time quantitative PCR in ovary and uterus of sows across pregnancy.

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María Martínez-Giner

Full Text Available BACKGROUND: Reproductive traits play a key role in pig production in order to reduce costs and increase economic returns. Among others, gene expression analyses represent a useful approach to study genetic mechanisms underlying reproductive traits in pigs. The application of reverse-transcription quantitative PCR requires the selection of appropriate reference genes, whose expression levels should not be affected by the experimental conditions, especially when comparing gene expression across different physiological stages. RESULTS: The gene expression stability of ten potential reference genes was studied by three different methods (geNorm, NormFinder and BestKeeper in ovary and uterus collected at five different physiological time points (heat, and 15, 30, 45 and 60 days of pregnancy. Although final ranking differed, the three algorithms gave very similar results. Thus, the most stable genes across time were TBP and UBC in uterus and TBP and HPRT1 in ovary, while HMBS and ACTB showed the less stable expression in uterus and ovary, respectively. When studied as a systematic effect, the reproductive stage did not significantly affect the expression of the candidate reference genes except at 30d and 60d of pregnancy, when a general drop in expression was observed in ovary. CONCLUSIONS: Based in our results, we propose the use of TBP, UBC and SDHA in uterus and TBP, GNB2L1 and HPRT1 in ovary for normalization of longitudinal expression studies using quantitative PCR in sows.

Study of differential gene expression in human hepatocyte exposed to 50 cGy γ ray

International Nuclear Information System (INIS)

Wen Jianhua; Li Jianguo; Tian Huancheng; Li Yanling; Wang Xiaoli; Zuo Yanhui

2008-01-01

The study analyzed the differential transcriptional profile of the normal human hepatic cell and the human hepatic cell radiated with 50 cGy γ ray by gene chip technique. The results showed that there were 614 differentially expressed genes among 14 112 human genes analyzed, in which 521 genes were up-regulated and 93 genes down-regulated. These genes are associated with mitochondrial regulation, homo sapiens hepatitis A virus cellular receptor, tumor necrosis factor, cell cycle regulation, kinase and zinc finger protein etc. RT-PCR results indicated that up-regulated expression of gene HAVcr-1, HAVcr-2, MFTC, MOAP1 and down-regulated expression of gene TRIP12, DCN were consistent with gene chip data. (authors)

SPAM1 (PH-20 protein and mRNA expression in the epididymides of humans and macaques: utilizing laser microdissection/RT-PCR

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Zhang Hong

2003-08-01

Full Text Available Abstract Background The Sperm Adhesion Molecule 1 (SPAM1 is an important sperm surface hyaluronidase with at least three functions in mammalian fertilization. Previously our laboratory reported that in the mouse, in addition to its expression in the testis, Spam1 is synthesized in the epididymis where it is found in membranous vesicles in the principal cells of the epithelium in all three regions. Since SPAM1 is widely conserved among mammals the aim of the study was to determine if its expression pattern in the epididymis is conserved in rodents and primates. Methods We used laser microdissection (LM/RT-PCR on frozen and paraffin-embedded epididymal sections of humans (n = 3 and macaques (n = 2 as well as in situ transcript hybridization to determine if transcripts are present in the epididymal epithelium. Western analysis and immunohistochemistry were used to detect and confirm the protein expression, and hyaluronic acid substrate gel electrophoresis analyzed its hyaluronidase activity. An in silico analysis of the proximal promoter of SPAM1 was also performed to identify relevant putative transcription binding sites for the androgen receptor. Results We demonstrate that mRNA unique to SPAM1 is present in the principal cells of the epididymal epithelium in all individuals of both species studied. SPAM1 protein is present in all three regions of the epididymis, as well as the vas deferens, and is localized similarly to the transcripts. SPAM1 was shown to have hyaluronidase activity at pH 7.0. In the proximal promoter of SPAM1 were uncovered putative epididymal transcription factor binding sites including androgen receptor elements (AREs, consistent with epididymal expression. Conclusions These findings allow us to conclude that epididymal SPAM1 is conserved in at least two mammalian classes, rodents and primates. This conservation of expression suggests that the protein is likely to play an important function, possibly in sperm maturation.

PCR

African Journals Online (AJOL)

Elham

2013-07-03

Jul 3, 2013 ... was constructed with competitive strategy by PCR-cloning technique and the limitation range was determined. The PCR products of MTB and IAC were 245 and 660 bp, respectively on .... products' differentiation was easy.

Evaluation of endogenous control gene(s) for gene expression studies in human blood exposed to 60Co γ-rays ex vivo.

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Vaiphei, S Thangminlal; Keppen, Joshua; Nongrum, Saibadaiahun; Chaubey, R C; Kma, L; Sharan, R N

2015-01-01

In gene expression studies, it is critical to normalize data using a stably expressed endogenous control gene in order to obtain accurate and reliable results. However, we currently do not have a universally applied endogenous control gene for normalization of data for gene expression studies, particularly those involving (60)Co γ-ray-exposed human blood samples. In this study, a comparative assessment of the gene expression of six widely used housekeeping endogenous control genes, namely 18S, ACTB, B2M, GAPDH, MT-ATP6 and CDKN1A, was undertaken for a range of (60)Co γ-ray doses (0.5, 1.0, 2.0 and 4.0 Gy) at 8.4 Gy min(-1) at 0 and 24 h post-irradiation time intervals. Using the NormFinder algorithm, real-time PCR data obtained from six individuals (three males and three females) were analyzed with respect to the threshold cycle (Ct) value and abundance, ΔCt pair-wise comparison, intra- and inter-group variability assessments, etc. GAPDH, either alone or in combination with 18S, was found to be the most suitable endogenous control gene and should be used in gene expression studies, especially those involving qPCR of γ-ray-exposed human blood samples. © The Author 2014. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

Usefullness of IGH/TCR PCR studies in lymphoproliferative disorders with inconclusive clonality by flow cytometry.

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Ribera, Jordi; Zamora, Lurdes; Juncà, Jordi; Rodríguez, Inés; Marcé, Silvia; Cabezón, Marta; Millá, Fuensanta

2013-07-25

In up to 5-15% of studies of lymphoproliferative disorders (LPD) flow cytometry (FCM) or immunomorphologic methods cannot discriminate malignant from reactive processes. The aim of this work was to determine the usefulness of PCR for solving these diagnostic uncertainties. We analyzed IGH and TCRγ genes by PCR in 106 samples with inconclusive FCM results. A clonal result was registered in 36/106 studies, with a LPD being confirmed in 27 (75%) of these cases. Specifically, 9/9 IGH clonal and 16/25 TCRγ clonal results were finally diagnosed with LPD. Additionally, 2 clonal TCRγ samples with suspicion of undefined LPD were finally diagnosed with T LPD. Although polyclonal results were obtained in 47 of the cases studied (38 IGH and 9 TCRγ), hematologic neoplasms were diagnosed in 4/38 IGH polyclonal and in 1/9 TCRγ polyclonal studies. There were also 14 PCR polyclonal results (4 IGH, 10 TCRγ), albeit non-conclusive. Of these, 2/4 were eventually diagnosed with B-cell lymphoma and 3/10 with T-cell LPD. In 8 IGH samples the results of PCR techniques were non-informative but in 3/8 cases a B lymphoma was finally confirmed. We concluded that PCR is a useful technique to identify LPD when FCM is inconclusive. A PCR clonal B result is indicative of malignancy but IGH polyclonal and non-conclusive results do not exclude lymphoid neoplasms. Interpretation of T-cell clonality should be based on all the available clinical and analytical data. © 2013 Clinical Cytometry Society. Copyright © 2013 Clinical Cytometry Society.

In vitro and in vivo MMP gene expression localisation by In Situ-RT-PCR in cell culture and paraffin embedded human breast cancer cell line xenografts

International Nuclear Information System (INIS)

Haupt, Larisa M; Thompson, Erik W; Trezise, Ann EO; Irving, Rachel E; Irving, Michael G; Griffiths, Lyn R

2006-01-01

Members of the matrix metalloproteinase (MMP) family of proteases are required for the degradation of the basement membrane and extracellular matrix in both normal and pathological conditions. In vitro, MT1-MMP (MMP-14, membrane type-1-MMP) expression is higher in more invasive human breast cancer (HBC) cell lines, whilst in vivo its expression has been associated with the stroma surrounding breast tumours. MMP-1 (interstitial collagenase) has been associated with MDA-MB-231 invasion in vitro, while MMP-3 (stromelysin-1) has been localised around invasive cells of breast tumours in vivo. As MMPs are not stored intracellularly, the ability to localise their expression to their cells of origin is difficult. We utilised the unique in situ-reverse transcription-polymerase chain reaction (IS-RT-PCR) methodology to localise the in vitro and in vivo gene expression of MT1-MMP, MMP-1 and MMP-3 in human breast cancer. In vitro, MMP induction was examined in the MDA-MB-231 and MCF-7 HBC cell lines following exposure to Concanavalin A (Con A). In vivo, we examined their expression in archival paraffin embedded xenografts derived from a range of HBC cell lines of varied invasive and metastatic potential. Mouse xenografts are heterogenous, containing neoplastic human parenchyma with mouse stroma and vasculature and provide a reproducible in vivo model system correlated to the human disease state. In vitro, exposure to Con A increased MT1-MMP gene expression in MDA-MB-231 cells and decreased MT1-MMP gene expression in MCF-7 cells. MMP-1 and MMP-3 gene expression remained unchanged in both cell lines. In vivo, stromal cells recruited into each xenograft demonstrated differences in localised levels of MMP gene expression. Specifically, MDA-MB-231, MDA-MB-435 and Hs578T HBC cell lines are able to influence MMP gene expression in the surrounding stroma. We have demonstrated the applicability and sensitivity of IS-RT-PCR for the examination of MMP gene expression both in vitro and in

Identification of Suitable Reference Genes for Investigating Gene Expression in Anterior Cruciate Ligament Injury by Using Reverse Transcription-Quantitative PCR.

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Mariana Ferreira Leal

Full Text Available The anterior cruciate ligament (ACL is one of the most frequently injured structures during high-impact sporting activities. Gene expression analysis may be a useful tool for understanding ACL tears and healing failure. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR has emerged as an effective method for such studies. However, this technique requires the use of suitable reference genes for data normalization. Here, we evaluated the suitability of six reference genes (18S, ACTB, B2M, GAPDH, HPRT1, and TBP by using ACL samples of 39 individuals with ACL tears (20 with isolated ACL tears and 19 with ACL tear and combined meniscal injury and of 13 controls. The stability of the candidate reference genes was determined by using the NormFinder, geNorm, BestKeeper DataAssist, and RefFinder software packages and the comparative ΔCt method. ACTB was the best single reference gene and ACTB+TBP was the best gene pair. The GenEx software showed that the accumulated standard deviation is reduced when a larger number of reference genes is used for gene expression normalization. However, the use of a single reference gene may not be suitable. To identify the optimal combination of reference genes, we evaluated the expression of FN1 and PLOD1. We observed that at least 3 reference genes should be used. ACTB+HPRT1+18S is the best trio for the analyses involving isolated ACL tears and controls. Conversely, ACTB+TBP+18S is the best trio for the analyses involving (1 injured ACL tears and controls, and (2 ACL tears of patients with meniscal tears and controls. Therefore, if the gene expression study aims to compare non-injured ACL, isolated ACL tears and ACL tears from patients with meniscal tear as three independent groups ACTB+TBP+18S+HPRT1 should be used. In conclusion, 3 or more genes should be used as reference genes for analysis of ACL samples of individuals with and without ACL tears.

Gene expression of inflammation and bone healing in peri-implant crevicular fluid after placement and loading of dental implants. A kinetic clinical pilot study using quantitative real-time PCR.

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Slotte, Christer; Lennerås, Maria; Göthberg, Catharina; Suska, Felicia; Zoric, Neven; Thomsen, Peter; Nannmark, Ulf

2012-10-01

Early detection of healing complications after placement of dental implants is a pressing but elusive goal. This paper proposes a non-invasive diagnostic tool for monitoring healing- and peri-implant disease specific genes, complementary to clinical evaluations. Eighteen partially edentulous patients were recruited to this pilot study. Three Brånemark TiUnite® implants/patient (Nobel Biocare) were placed in a one-stage procedure. Abutments with smooth or rough (TiUnite®) surface were placed. The test group (n = 9) received fixed bridges (immediate loading), whereas the control group (n = 9) implants were loaded 3 months after surgery. In addition to clinical measurements, crevicular fluid was collected using paper strips at the implant abutments 2, 14, 28, and 90 days postoperative. mRNA was extracted, purified, and converted to cDNA. Quantitative PCR assays for IL-1β, TNF-α, Osteocalcin (OC), Alkaline Phosphatase (ALP), Cathepsin K, Tartrate Resistant Acid Phosphatase, and 18S ribosomal RNA were designed and validated. Relative gene expression levels were calculated. One implant was lost in the control group and three in the test group. In one test patient, one implant showed lowered stability after 2 to 4 weeks and was unloaded. Later implant stability improved which allowed for loading after 3 to 4 months. TNF-α and ALP most commonly showed correlation with clinical parameters followed by IL-1β and OC. The strongest correlation was found for TNF-α with clinical complications at 2 and 14 days (p = .01/r = -048, and p = .0004/r = -0.56, respectively; test and control groups together). In some cases, gene expression predicted clinical complications (TNF-α, ALP, CK). This study is based on samples from few individuals; still, some genes showed correlation with clinical findings. Further studies are needed to refine and optimize the sampling process, to find the appropriate panel, and to validate gene expression for monitoring implant healing. © 2010 Wiley

Comparison of allele-specific PCR, created restriction-site PCR, and PCR with primer-introduced restriction analysis methods used for screening complex vertebral malformation carriers in Holstein cattle

Science.gov (United States)

Altınel, Ahmet

2017-01-01

Complex vertebral malformation (CVM) is an inherited, autosomal recessive disorder of Holstein cattle. The aim of this study was to compare sensitivity, specificity, positive and negative predictive values, accuracy, and rapidity of allele-specific polymerase chain reaction (AS-PCR), created restriction-site PCR (CRS-PCR), and PCR with primer-introduced restriction analysis (PCR-PIRA), three methods used in identification of CVM carriers in a Holstein cattle population. In order to screen for the G>T mutation in the solute carrier family 35 member A3 (SLC35A3) gene, DNA sequencing as the gold standard method was used. The prevalence of carriers and the mutant allele frequency were 3.2% and 0.016, respectively, among Holstein cattle in the Thrace region of Turkey. Among the three methods, the fastest but least accurate was AS-PCR. Although the rapidity of CRS-PCR and PCR-PIRA were nearly equal, the accuracy of PCR-PIRA was higher than that of CRS-PCR. Therefore, among the three methods, PCR-PIRA appears to be the most efficacious for screening of mutant alleles when identifying CVM carriers in a Holstein cattle population. PMID:28927256

Tissue-specific Calibration of Real-time PCR Facilitates Absolute Quantification of Plasmid DNA in Biodistribution Studies

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Joan K Ho

2016-01-01

Full Text Available Analysis of the tissue distribution of plasmid DNA after administration of nonviral gene delivery systems is best accomplished using quantitative real-time polymerase chain reaction (qPCR, although published strategies do not allow determination of the absolute mass of plasmid delivered to different tissues. Generally, data is expressed as the mass of plasmid relative to the mass of genomic DNA (gDNA in the sample. This strategy is adequate for comparisons of efficiency of delivery to a single site but it does not allow direct comparison of delivery to multiple tissues, as the mass of gDNA extracted per unit mass of each tissue is different. We show here that by constructing qPCR standard curves for each tissue it is possible to determine the dose of intact plasmid remaining in each tissue, which is a more useful parameter when comparing the fates of different formulations of DNA. We exemplify the use of this tissue-specific qPCR method by comparing the delivery of naked DNA, cationic DNA complexes, and neutral PEGylated DNA complexes after intramuscular injection. Generally, larger masses of intact plasmid were present 24 hours after injection of DNA complexes, and neutral complexes resulted in delivery of a larger mass of intact plasmid to the spleen.

Identification and Evaluation of Reliable Reference Genes for Quantitative Real-Time PCR Analysis in Tea Plant (Camellia sinensis (L.) O. Kuntze)

Science.gov (United States)

Hao, Xinyuan; Horvath, David P.; Chao, Wun S.; Yang, Yajun; Wang, Xinchao; Xiao, Bin

2014-01-01

Reliable reference selection for the accurate quantification of gene expression under various experimental conditions is a crucial step in qRT-PCR normalization. To date, only a few housekeeping genes have been identified and used as reference genes in tea plant. The validity of those reference genes are not clear since their expression stabilities have not been rigorously examined. To identify more appropriate reference genes for qRT-PCR studies on tea plant, we examined the expression stability of 11 candidate reference genes from three different sources: the orthologs of Arabidopsis traditional reference genes and stably expressed genes identified from whole-genome GeneChip studies, together with three housekeeping gene commonly used in tea plant research. We evaluated the transcript levels of these genes in 94 experimental samples. The expression stabilities of these 11 genes were ranked using four different computation programs including geNorm, Normfinder, BestKeeper, and the comparative ∆CT method. Results showed that the three commonly used housekeeping genes of CsTUBULIN1, CsACINT1 and Cs18S rRNA1 together with CsUBQ1 were the most unstable genes in all sample ranking order. However, CsPTB1, CsEF1, CsSAND1, CsCLATHRIN1 and CsUBC1 were the top five appropriate reference genes for qRT-PCR analysis in complex experimental conditions. PMID:25474086

The construction and use of bacterial DNA microarrays based on an optimized two-stage PCR strategy

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Pesta David

2003-06-01

Full Text Available Abstract Background DNA microarrays are a powerful tool with important applications such as global gene expression profiling. Construction of bacterial DNA microarrays from genomic sequence data using a two-stage PCR amplification approach for the production of arrayed DNA is attractive because it allows, in principal, the continued re-amplification of DNA fragments and facilitates further utilization of the DNA fragments for additional uses (e.g. over-expression of protein. We describe the successful construction and use of DNA microarrays by the two-stage amplification approach and discuss the technical challenges that were met and resolved during the project. Results Chimeric primers that contained both gene-specific and shared, universal sequence allowed the two-stage amplification of the 3,168 genes identified on the genome of Synechocystis sp. PCC6803, an important prokaryotic model organism for the study of oxygenic photosynthesis. The gene-specific component of the primer was of variable length to maintain uniform annealing temperatures during the 1st round of PCR synthesis, and situated to preserve full-length ORFs. Genes were truncated at 2 kb for efficient amplification, so that about 92% of the PCR fragments were full-length genes. The two-stage amplification had the additional advantage of normalizing the yield of PCR products and this improved the uniformity of DNA features robotically deposited onto the microarray surface. We also describe the techniques utilized to optimize hybridization conditions and signal-to-noise ratio of the transcription profile. The inter-lab transportability was demonstrated by the virtual error-free amplification of the entire genome complement of 3,168 genes using the universal primers in partner labs. The printed slides have been successfully used to identify differentially expressed genes in response to a number of environmental conditions, including salt stress. Conclusions The technique detailed

Absolute quantification by droplet digital PCR versus analog real-time PCR

Science.gov (United States)

Hindson, Christopher M; Chevillet, John R; Briggs, Hilary A; Gallichotte, Emily N; Ruf, Ingrid K; Hindson, Benjamin J; Vessella, Robert L; Tewari, Muneesh

2014-01-01

Nanoliter-sized droplet technology paired with digital PCR (ddPCR) holds promise for highly precise, absolute nucleic acid quantification. Our comparison of microRNA quantification by ddPCR and real-time PCR revealed greater precision (coefficients of variation decreased by 37–86%) and improved day-to-day reproducibility (by a factor of seven) of ddPCR but with comparable sensitivity. When we applied ddPCR to serum microRNA biomarker analysis, this translated to superior diagnostic performance for identifying individuals with cancer. PMID:23995387

Comparison of a Commercially Available Repetitive-Element PCR System (DiversiLab) with PCR Ribotyping for Typing of Clostridium difficile Strains ▿

OpenAIRE

Eckert, C.; Van Broeck, J.; Spigaglia, P.; Burghoffer, B.; Delmée, M.; Mastrantonio, P.; Barbut, F.

2011-01-01

This study compared a repetitive-element PCR (rep-PCR) method (DiversiLab system) to PCR ribotyping. The discriminatory power of rep-PCR was 0.997. Among the PCR ribotype 027 isolates tested, different rep types could be distinguished. rep-PCR showed a higher discriminatory power than PCR ribotyping. Nevertheless, this method requires technical skill, and visual interpretation of rep-PCR fingerprint patterns may be difficult.

Selection and validation of endogenous reference genes for qRT-PCR analysis in leafy spurge (Euphorbia esula.

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Wun S Chao

Full Text Available Quantitative real-time polymerase chain reaction (qRT-PCR is the most important tool in measuring levels of gene expression due to its accuracy, specificity, and sensitivity. However, the accuracy of qRT-PCR analysis strongly depends on transcript normalization using stably expressed reference genes. The aim of this study was to find internal reference genes for qRT-PCR analysis in various experimental conditions for seed, adventitious underground bud, and other organs of leafy spurge. Eleven candidate reference genes (BAM4, PU1, TRP-like, FRO1, ORE9, BAM1, SEU, ARF2, KAPP, ZTL, and MPK4 were selected from among 171 genes based on expression stabilities during seed germination and bud growth. The other ten candidate reference genes were selected from three different sources: (1 3 stably expressed leafy spurge genes (60S, bZIP21, and MD-100 identified from the analyses of leafy spurge microarray data; (2 3 orthologs of Arabidopsis "general purpose" traditional reference genes (GAPDH_1, GAPDH_2, and UBC; and (3 4 orthologs of Arabidopsis stably expressed genes (UBC9, SAND, PTB, and F-box identified from Affymetrix ATH1 whole-genome GeneChip studies. The expression stabilities of these 21 genes were ranked based on the C(T values of 72 samples using four different computation programs including geNorm, Normfinder, BestKeeper, and the comparative ΔC(T method. Our analyses revealed SAND, PTB, ORE9, and ARF2 to be the most appropriate reference genes for accurate normalization of gene expression data. Since SAND and PTB were obtained from 4 orthologs of Arabidopsis, while ORE9 and ARF2 were selected from 171 leafy spurge genes, it was more efficient to identify good reference genes from the orthologs of other plant species that were known to be stably expressed than that of randomly testing endogenous genes. Nevertheless, the two newly identified leafy spurge genes, ORE9 and ARF2, can serve as orthologous candidates in the search for reference genes

Screening suitable reference genes for normalization in reverse transcription quantitative real-time PCR analysis in melon.

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Qiusheng Kong

Full Text Available Melon (Cucumis melo. L is not only an economically important cucurbitaceous crop but also an attractive model for studying many biological characteristics. Screening appropriate reference genes is essential to reverse transcription quantitative real-time PCR (RT-qPCR, which is key to many studies involving gene expression analysis. In this study, 14 candidate reference genes were selected, and the variations in their expression in roots and leaves of plants subjected to biotic stress, abiotic stress, and plant growth regulator treatment were assessed by RT-qPCR. The stability of the expression of the selected genes was determined and ranked using geNorm and NormFinder. geNorm identified the two most stable genes for each set of conditions: CmADP and CmUBIep across all samples, CmUBIep and CmRPL in roots, CmRAN and CmACT in leaves, CmADP and CmRPL under abiotic stress conditions, CmTUA and CmACT under biotic stress conditions, and CmRAN and CmACT under plant growth regulator treatments. NormFinder determined CmRPL to be the best reference gene in roots and under biotic stress conditions and CmADP under the other experimental conditions. CmUBC2 and CmPP2A were not found to be suitable under many experimental conditions. The catalase family genes CmCAT1, CmCAT2, and CmCAT3 were identified in melon genome and used as target genes to validate the reliability of identified reference genes. The catalase family genes showed the most upregulation 3 days after inoculation with Fusarium wilt in roots, after which they were downregulated. Their levels of expression were significantly overestimated when the unsuitable reference gene was used for normalization. These results not only provide guidelines for the selection of reference genes for gene expression analyses in melons but may also provide valuable information for studying the functions of catalase family genes in stress responses.

Method Enabling Gene Expression Studies of Pathogens in a Complex Food Matrix

DEFF Research Database (Denmark)

Kjeldgaard, Jette; Henriksen, Sidsel; Cohn, Marianne Thorup

2011-01-01

We describe a simple method for stabilizing and extracting high-quality prokaryotic RNA from meat. Heat and salt stress of Escherichia coli and Salmonella spp. in minced meat reproducibly induced dnaK and otsB expression, respectively, as observed by quantitative reverse transcription-PCR (>5-fold...

Evaluation of Housekeeping Genes for Quantitative Real-Time PCR Analysis of Bradysia odoriphaga (Diptera: Sciaridae

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Caihua Shi

2016-07-01

Full Text Available The soil insect Bradysia odoriphaga (Diptera: Sciaridae causes substantial damage to Chinese chive. Suitable reference genes in B. odoriphaga (Bradysia odoriphaga have yet to be identified for normalizing target gene expression among samples by quantitative real-time PCR (qRT-PCR. This study was focused on identifying the expression stability of 12 candidate housekeeping genes in B. odoriphaga under various experiment conditions. The final stability ranking of 12 housekeeping genes was obtained with RefFinder, and the most suitable number of reference genes was analyzed by GeNorm. The results revealed that the most appropriate sets of internal controls were RPS15, RPL18, and RPS18 across developmental phases; RPS15, RPL28, and GAPDH across temperatures; RPS15 and RPL18 across pesticide treatments; RSP5, RPS18, and SDHA across photoperiods; ACTb, RPS18, and RPS15 across diets; RPS13 and RPL28 across populations; and RPS15, ACTb, and RPS18 across all samples. The use of the most suitable reference genes versus an arbitrarily selected reference gene resulted in significant differences in the analysis of a target gene expression. HSP23 in B. odoriphaga was found to be up-regulated under low temperatures. These results will contribute to the standardization of qRT-PCR and will also be valuable for further research on gene function in B. odoriphaga.

Recommended Reference Genes for Quantitative PCR Analysis in Soybean Have Variable Stabilities during Diverse Biotic Stresses.

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Raman Bansal

Full Text Available For real-time reverse transcription-PCR (qRT-PCR in soybean, reference genes in different tissues, developmental stages, various cultivars, and under stress conditions have been suggested but their usefulness for research on soybean under various biotic stresses occurring in North-Central U.S. is not known. Here, we investigated the expression stabilities of ten previously recommended reference genes (ABCT, CYP, EF1A, FBOX, GPDH, RPL30, TUA4, TUB4, TUA5, and UNK2 in soybean under biotic stress from Bean pod mottle virus (BPMV, powdery mildew (PMD, soybean aphid (SBA, and two-spotted spider mite (TSSM. BPMV, PMD, SBA, and TSSM are amongst the most common pest problems on soybean in North-Central U.S. and other regions. Reference gene stability was determined using three software algorithms (geNorm, NormFinder, BestKeeper and a web-based tool (RefFinder. Reference genes showed variability in their expression as well as stability across various stressors and the best reference genes were stress-dependent. ABCT and FBOX were found to be the most stable in soybean under both BPMV and SBA stress but these genes had only minimal to moderate stability during PMD and TSSM stress. Expression of TUA4 and CYP was found to be most stable during PMD stress; TUB4 and TUA4 were stable under TSSM stress. Under various biotic stresses on soybean analyzed, GPDH expression was found to be consistently unstable. For all biotic stressors on soybean, we obtained pairwise variation (V2/3 values less than 0.15 which suggested that combined use of the two most stable reference genes would be sufficient for normalization. Further, we demonstrated the utility of normalizing the qRT-PCR data for target genes using the most stable reference genes validated in current study. Following of the recommendations from our current study will enable an accurate and reliable normalization of qRT-PCR data in soybean under biotic stress.

Applications of the rep-PCR DNA fingerprinting technique to study microbial diversity, ecology and evolution.

Science.gov (United States)

Ishii, Satoshi; Sadowsky, Michael J

2009-04-01

A large number of repetitive DNA sequences are found in multiple sites in the genomes of numerous bacteria, archaea and eukarya. While the functions of many of these repetitive sequence elements are unknown, they have proven to be useful as the basis of several powerful tools for use in molecular diagnostics, medical microbiology, epidemiological analyses and environmental microbiology. The repetitive sequence-based PCR or rep-PCR DNA fingerprint technique uses primers targeting several of these repetitive elements and PCR to generate unique DNA profiles or 'fingerprints' of individual microbial strains. Although this technique has been extensively used to examine diversity among variety of prokaryotic microorganisms, rep-PCR DNA fingerprinting can also be applied to microbial ecology and microbial evolution studies since it has the power to distinguish microbes at the strain or isolate level. Recent advancement in rep-PCR methodology has resulted in increased accuracy, reproducibility and throughput. In this minireview, we summarize recent improvements in rep-PCR DNA fingerprinting methodology, and discuss its applications to address fundamentally important questions in microbial ecology and evolution.

Application of droplet digital PCR for quantitative detection of Spiroplasma citri in comparison with real time PCR.

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Yogita Maheshwari

Full Text Available Droplet digital polymerase chain reaction (ddPCR is a method for performing digital PCR that is based on water-oil emulsion droplet technology. It is a unique approach to measure the absolute copy number of nucleic acid targets without the need of external standards. This study evaluated the applicability of ddPCR as a quantitative detection tool for the Spiroplasma citri, causal agent of citrus stubborn disease (CSD in citrus. Two sets of primers, SP1, based on the spiral in housekeeping gene, and a multicopy prophage gene, SpV1 ORF1, were used to evaluate ddPCR in comparison with real time (quantitative PCR (qPCR for S. citri detection in citrus tissues. Standard curve analyses on tenfold dilution series showed that both ddPCR and qPCR exhibited good linearity and efficiency. However, ddPCR had a tenfold greater sensitivity than qPCR and accurately quantified up to one copy of spiralin gene. Receiver operating characteristic analysis indicated that the ddPCR methodology was more robust for diagnosis of CSD and the area under the curve was significantly broader compared to qPCR. Field samples were used to validate ddPCR efficacy and demonstrated that it was equal or better than qPCR to detect S. citri infection in fruit columella due to a higher pathogen titer. The ddPCR assay detected both the S. citri spiralin and the SpV1 ORF1 targets quantitatively with high precision and accuracy compared to qPCR assay. The ddPCR was highly reproducible and repeatable for both the targets and showed higher resilience to PCR inhibitors in citrus tissue extract for the quantification of S. citri compare to qPCR.

18S rRNA is a reliable normalisation gene for real time PCR based on influenza virus infected cells

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Kuchipudi Suresh V

2012-10-01

Full Text Available Abstract Background One requisite of quantitative reverse transcription PCR (qRT-PCR is to normalise the data with an internal reference gene that is invariant regardless of treatment, such as virus infection. Several studies have found variability in the expression of commonly used housekeeping genes, such as beta-actin (ACTB and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, under different experimental settings. However, ACTB and GAPDH remain widely used in the studies of host gene response to virus infections, including influenza viruses. To date no detailed study has been described that compares the suitability of commonly used housekeeping genes in influenza virus infections. The present study evaluated several commonly used housekeeping genes [ACTB, GAPDH, 18S ribosomal RNA (18S rRNA, ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide (ATP5B and ATP synthase, H+ transporting, mitochondrial Fo complex, subunit C1 (subunit 9 (ATP5G1] to identify the most stably expressed gene in human, pig, chicken and duck cells infected with a range of influenza A virus subtypes. Results The relative expression stability of commonly used housekeeping genes were determined in primary human bronchial epithelial cells (HBECs, pig tracheal epithelial cells (PTECs, and chicken and duck primary lung-derived cells infected with five influenza A virus subtypes. Analysis of qRT-PCR data from virus and mock infected cells using NormFinder and BestKeeper software programmes found that 18S rRNA was the most stable gene in HBECs, PTECs and avian lung cells. Conclusions Based on the presented data from cell culture models (HBECs, PTECs, chicken and duck lung cells infected with a range of influenza viruses, we found that 18S rRNA is the most stable reference gene for normalising qRT-PCR data. Expression levels of the other housekeeping genes evaluated in this study (including ACTB and GPADH were highly affected by influenza virus infection and

Identification of endogenous control genes for normalisation of real-time quantitative PCR data in colorectal cancer.

LENUS (Irish Health Repository)

Kheirelseid, Elrasheid A H

2010-01-01

BACKGROUND: Gene expression analysis has many applications in cancer diagnosis, prognosis and therapeutic care. Relative quantification is the most widely adopted approach whereby quantification of gene expression is normalised relative to an endogenously expressed control (EC) gene. Central to the reliable determination of gene expression is the choice of control gene. The purpose of this study was to evaluate a panel of candidate EC genes from which to identify the most stably expressed gene(s) to normalise RQ-PCR data derived from primary colorectal cancer tissue. RESULTS: The expression of thirteen candidate EC genes: B2M, HPRT, GAPDH, ACTB, PPIA, HCRT, SLC25A23, DTX3, APOC4, RTDR1, KRTAP12-3, CHRNB4 and MRPL19 were analysed in a cohort of 64 colorectal tumours and tumour associated normal specimens. CXCL12, FABP1, MUC2 and PDCD4 genes were chosen as target genes against which a comparison of the effect of each EC gene on gene expression could be determined. Data analysis using descriptive statistics, geNorm, NormFinder and qBasePlus indicated significant difference in variances between candidate EC genes. We determined that two genes were required for optimal normalisation and identified B2M and PPIA as the most stably expressed and reliable EC genes. CONCLUSION: This study identified that the combination of two EC genes (B2M and PPIA) more accurately normalised RQ-PCR data in colorectal tissue. Although these control genes might not be optimal for use in other cancer studies, the approach described herein could serve as a template for the identification of valid ECs in other cancer types.

Pneumocystis PCR: It Is Time to Make PCR the Test of Choice.

Science.gov (United States)

Doyle, Laura; Vogel, Sherilynn; Procop, Gary W

2017-01-01

The testing strategy for Pneumocystis at the Cleveland Clinic changed from toluidine blue staining to polymerase chain reaction (PCR). We studied the differences in positivity rates for these assays and compared each with the detection of Pneumocystis in companion specimens by cytology and surgical pathology. We reviewed the results of all Pneumocystis test orders 1 year before and 1 year after the implementation of a Pneumocystis -specific PCR. We also reviewed the corresponding cytology and surgical pathology results, if performed. Finally, we reviewed the medical records of patients with rare Pneumocystis detected by PCR in an effort to differentiate colonization vs true disease. Toluidine blue staining and surgical pathology had similar sensitivities and negative predictive values, both of which were superior to cytology. There was a >4-fold increase in the annual detection of Pneumocystis by PCR compared with toluidine blue staining (toluidine blue staining: 11/1583 [0.69%] vs PCR: 44/1457 [3.0%]; chi-square P < .001). PCR detected 1 more case than surgical pathology and was far more sensitive than cytology. Chart review demonstrated that the vast majority of patients with rare Pneumocystis detected were immunosuppressed, had radiologic findings supportive of this infection, had no other pathogens detected, and were treated for pneumocystosis by the clinical team. PCR was the most sensitive method for the detection of Pneumocystis and should be considered the diagnostic test of choice. Correlation with clinical and radiologic findings affords discrimination of early true disease from the far rarer instances of colonization.

Eliminating PCR contamination

International Nuclear Information System (INIS)

Fox, J.C.; Ait-Khaled, Mounir; Webster, Alison; Emery, V.C.

1991-01-01

The sensitivity of polymerase chain reaction (PCR) can mean that even very low levels of contamination with the target DNA will result in a positive signal. At present this aspect is a major limitation in the use of PCR as a routine diagnostic method. By exposing PCR reagents to UV light, contaminating DNA can be inactivated, thus providing an opportunity to eradicate false positive reactions. UV irradiation was applied to PCR systems used for detection of human cytomegalovirus CMV and human immunodeficiency virus (HIV) and shown to be effective in eradicating both laboratory encountered contamination and plasmid DNA (below 100 pg) added to PCR systems prior to UV exposure. Sensitivity of a PCR system to amplify the long terminal repeat (LTR) sequence of HIV-1 was not affected by the irradiation procedure; however, ultimate sensitivity of a PCR system for the amplification of an early gene pro-motor sequence of the CMV genome was reduced 1000-fold. UV irradiation did not affect the size of the PCR product as determined by strand separating polyacrylamide gel electrophoresis of a 32 P-labelled amplimer. Thus, a simple pre-exposure to UV light would seem a worth-wile step to incorporate into PCR protocols provided that the effects on sensitivity have been determined empirically for each PCR system. (author). 11 refs.; 3 figs

A simple RT-PCR-based strategy for screening connexin identity

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M. Urban

1999-08-01

Full Text Available Vertebrate gap junctions are aggregates of transmembrane channels which are composed of connexin (Cx proteins encoded by at least fourteen distinct genes in mammals. Since the same Cx type can be expressed in different tissues and more than one Cx type can be expressed by the same cell, the thorough identification of which connexin is in which cell type and how connexin expression changes after experimental manipulation has become quite laborious. Here we describe an efficient, rapid and simple method by which connexin type(s can be identified in mammalian tissue and cultured cells using endonuclease cleavage of RT-PCR products generated from "multi primers" (sense primer, degenerate oligonucleotide corresponding to a region of the first extracellular domain; antisense primer, degenerate oligonucleotide complementary to the second extracellular domain that amplify the cytoplasmic loop regions of all known connexins except Cx36. In addition, we provide sequence information on RT-PCR primers used in our laboratory to screen individual connexins and predictions of extension of the "multi primer" method to several human connexins.

Droplet digital PCR analysis of NOTCH1 gene mutations in chronic lymphocytic leukemia.

Science.gov (United States)

Minervini, Angela; Francesco Minervini, Crescenzio; Anelli, Luisa; Zagaria, Antonella; Casieri, Paola; Coccaro, Nicoletta; Cumbo, Cosimo; Tota, Giuseppina; Impera, Luciana; Orsini, Paola; Brunetti, Claudia; Giordano, Annamaria; Specchia, Giorgina; Albano, Francesco

2016-12-27

In chronic lymphocytic leukemia (CLL), NOTCH1 gene mutations (NOTCH1mut) have been associated with adverse prognostic features but the independence of these as a prognostic factor is still controversial. In our study we validated a c.7541-7542delCT NOTCH1 mutation assay based on droplet digital PCR (ddPCR); we also analyzed the NOTCH1mut allelic burden, expressed as fractional abundance (FA), in 88 CLL patients at diagnosis to assess its prognostic role and made a longitudinal ddPCR analysis in 10 cases harboring NOTCH1mut to verify the FA variation over time. Our data revealed that with the ddPCR approach the incidence of NOTCH1mut in CLL was much higher (53.4%) than expected. However, longitudinal ddPCR analysis of CLL cases showed a statistically significant reduction of the NOTCH1mut FA detected at diagnosis after treatment (median FA 11.67 % vs 0.09 %, respectively, p = 0.01); the same difference, in terms of NOTCH1mut FA, was observed in the relapsed cases compared to the NOTCH1mut allelic fraction observed in patients in complete or partial remission (median FA 4.75% vs 0.43%, respectively, p = 0.007). Our study demonstrated a much higher incidence of NOTCH1mut in CLL than has previously been reported, and showed that the NOTCH1mut allelic burden evaluation by ddPCR might identify patients in need of a closer clinical follow-up during the "watch and wait" interval and after standard chemotherapy.

Comparative validation using quantitative real-time PCR (qPCR and conventional PCR of bovine semen centrifuged in continuous density gradient

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M.V. Resende

2011-06-01

Full Text Available The objective of the present study was to determine the sperm enrichment with X-bearing spermatozoa, after one centrifugation in a Percoll or OptiPrep continuous density gradient, using quantitative real-time polymerase chain reaction (qPCR of sperm DNA and resultant in vitro-produced bovine embryos by PCR. Frozen/thawed sperm was layered on density gradients and the tubes were centrifuged. Supernatants were gently aspirated and the sperm recovered from the bottom of the tubes. Cleavage and blastocyst rates were determined through in vitro production of embryos and PCR was performed to identify the embryos' genetic sex. A difference in blastocyst rate was found in the Percoll treatment compared to OptiPrep (P<0.05. The percentage of female embryos in the Percoll and OptiPrep groups was 62.0% and 47.1%, respectively. These results were confirmed by qPCR of spermatozoa DNA and underestimation was seen only in the Percoll group. It was possible to sexing sperm using simple approach.

MicroRNA expression profiling to identify and validate reference genes for relative quantification in colorectal cancer.

LENUS (Irish Health Repository)

Chang, Kah Hoong

2010-01-01

BACKGROUND: Advances in high-throughput technologies and bioinformatics have transformed gene expression profiling methodologies. The results of microarray experiments are often validated using reverse transcription quantitative PCR (RT-qPCR), which is the most sensitive and reproducible method to quantify gene expression. Appropriate normalisation of RT-qPCR data using stably expressed reference genes is critical to ensure accurate and reliable results. Mi(cro)RNA expression profiles have been shown to be more accurate in disease classification than mRNA expression profiles. However, few reports detailed a robust identification and validation strategy for suitable reference genes for normalisation in miRNA RT-qPCR studies. METHODS: We adopt and report a systematic approach to identify the most stable reference genes for miRNA expression studies by RT-qPCR in colorectal cancer (CRC). High-throughput miRNA profiling was performed on ten pairs of CRC and normal tissues. By using the mean expression value of all expressed miRNAs, we identified the most stable candidate reference genes for subsequent validation. As such the stability of a panel of miRNAs was examined on 35 tumour and 39 normal tissues. The effects of normalisers on the relative quantity of established oncogenic (miR-21 and miR-31) and tumour suppressor (miR-143 and miR-145) target miRNAs were assessed. RESULTS: In the array experiment, miR-26a, miR-345, miR-425 and miR-454 were identified as having expression profiles closest to the global mean. From a panel of six miRNAs (let-7a, miR-16, miR-26a, miR-345, miR-425 and miR-454) and two small nucleolar RNA genes (RNU48 and Z30), miR-16 and miR-345 were identified as the most stably expressed reference genes. The combined use of miR-16 and miR-345 to normalise expression data enabled detection of a significant dysregulation of all four target miRNAs between tumour and normal colorectal tissue. CONCLUSIONS: Our study demonstrates that the top six most

MicroRNA expression profiling to identify and validate reference genes for relative quantification in colorectal cancer

LENUS (Irish Health Repository)

Chang, Kah Hoong

2010-04-29

Abstract Background Advances in high-throughput technologies and bioinformatics have transformed gene expression profiling methodologies. The results of microarray experiments are often validated using reverse transcription quantitative PCR (RT-qPCR), which is the most sensitive and reproducible method to quantify gene expression. Appropriate normalisation of RT-qPCR data using stably expressed reference genes is critical to ensure accurate and reliable results. Mi(cro)RNA expression profiles have been shown to be more accurate in disease classification than mRNA expression profiles. However, few reports detailed a robust identification and validation strategy for suitable reference genes for normalisation in miRNA RT-qPCR studies. Methods We adopt and report a systematic approach to identify the most stable reference genes for miRNA expression studies by RT-qPCR in colorectal cancer (CRC). High-throughput miRNA profiling was performed on ten pairs of CRC and normal tissues. By using the mean expression value of all expressed miRNAs, we identified the most stable candidate reference genes for subsequent validation. As such the stability of a panel of miRNAs was examined on 35 tumour and 39 normal tissues. The effects of normalisers on the relative quantity of established oncogenic (miR-21 and miR-31) and tumour suppressor (miR-143 and miR-145) target miRNAs were assessed. Results In the array experiment, miR-26a, miR-345, miR-425 and miR-454 were identified as having expression profiles closest to the global mean. From a panel of six miRNAs (let-7a, miR-16, miR-26a, miR-345, miR-425 and miR-454) and two small nucleolar RNA genes (RNU48 and Z30), miR-16 and miR-345 were identified as the most stably expressed reference genes. The combined use of miR-16 and miR-345 to normalise expression data enabled detection of a significant dysregulation of all four target miRNAs between tumour and normal colorectal tissue. Conclusions Our study demonstrates that the top six most

Foxp3 expression in human cancer cells

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Gourgoulianis Konstantinos I

2008-04-01

Full Text Available Abstract Objective Transcription factor forkhead box protein 3 (Foxp3 specifically characterizes the thymically derived naturally occurring regulatory T cells (Tregs. Limited evidence indicates that it is also expressed, albeit to a lesser extent, in tissues other than thymus and spleen, while, very recently, it was shown that Foxp3 is expressed by pancreatic carcinoma. This study was scheduled to investigate whether expression of Foxp3 transcripts and mature protein occurs constitutively in various tumor types. Materials and methods Twenty five tumor cell lines of different tissue origins (lung cancer, colon cancer, breast cancer, melanoma, erythroid leukemia, acute T-cell leukemia were studied. Detection of Foxp3 mRNA was performed using both conventional RT-PCR and quantitative real-time PCR while protein expression was assessed by immunocytochemistry and flow cytometry, using different antibody clones. Results Foxp3 mRNA as well as Foxp3 protein was detected in all tumor cell lines, albeit in variable levels, not related to the tissue of origin. This expression correlated with the expression levels of IL-10 and TGFb1. Conclusion We offer evidence that Foxp3 expression, characterizes tumor cells of various tissue origins. The biological significance of these findings warrants further investigation in the context of tumor immune escape, and especially under the light of current anti-cancer efforts interfering with Foxp3 expression.

Downregulation of Clusterin Expression in Human Testicular Seminoma

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Bianjiang Liu

2013-11-01

Full Text Available Background: Clusterin, a heterodimeric glycoprotein of approximately 80 kDa, exists extensively in human body fluids. The abnormal expression of clusterin is closely related to the occurrence, progression, and prognosis of tumors. Up to now, few studies have focused on clusterin in human testicular cancer. This study describes an extensive exploration of the presence and expression of clusterin in testicular seminoma. Methods: Tumor tissues and normal testis tissues were collected from 13 patients with testicular seminoma and 16 patients undergoing surgical castration for prostate cancer. Real-time polymerase chain reaction (PCR was performed to detect the expression difference of clusterin mRNA between testicular seminoma and normal testis. Western blot and immunohistochemical analysis were performed to detect the presence and expression difference of clusterin protein between two groups. Results: Real-time PCR showed the expression of clusterin mRNA in testicular seminoma to be significantly lower than in normal testis (only 13% relative quantification. Western blot analysis indicated marked reductions in the expression of clusterin protein in testicular seminoma. Similar results were observed upon immunohistochemical analysis. Conclusion: In testicular seminoma and normal testis, clusterin exists in its heterodimeric secretory isoform. Clusterin expression is significantly lower in testicular seminoma than in normal testis. This is the first comprehensive study of the presence and expression of clusterin in human testicular cancer.

Decreased expression of endogenous feline leukemia virus in cat lymphomas: a case control study.

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Krunic, Milica; Ertl, Reinhard; Hagen, Benedikt; Sedlazeck, Fritz J; Hofmann-Lehmann, Regina; von Haeseler, Arndt; Klein, Dieter

2015-04-10

Cats infected with exogenous feline leukemia virus (exFeLV) have a higher chance of lymphoma development than uninfected cats. Furthermore, an increased exFeLV transcription has been detected in lymphomas compared to non-malignant tissues. The possible mechanisms of lymphoma development by exFeLV are insertional mutagenesis or persistent stimulation of host immune cells by viral antigens, bringing them at risk for malignant transformation. Vaccination of cats against exFeLV has in recent years decreased the overall infection rate in most countries. Nevertheless, an increasing number of lymphomas have been diagnosed among exFeLV-negative cats. Endogenous feline leukemia virus (enFeLV) is another retrovirus for which transcription has been observed in cat lymphomas. EnFeLV provirus elements are present in the germline of various cat species and share a high sequence similarity with exFeLV but, due to mutations, are incapable of producing infectious viral particles. However, recombination between exFeLV and enFeLV could produce infectious particles. We examined the FeLV expression in cats that have developed malignant lymphomas and discussed the possible mechanisms that could have induced malignant transformation. For expression analysis we used next-generation RNA-sequencing (RNA-Seq) and for validation reverse transcription quantitative PCR (RT-qPCR). First, we showed that there was no expression of exFeLV in all samples, which eliminates the possibility of recombination between exFeLV and enFeLV. Next, we analyzed the difference in expression of three enFeLV genes between control and lymphoma samples. Our analysis showed an average of 3.40-fold decreased viral expression for the three genes in lymphoma compared to control samples. The results were confirmed by RT-qPCR. There is a decreased expression of enFeLV genes in lymphomas versus control samples, which contradicts previous observations for the exFeLV. Our results suggest that a persistent stimulation of host

Selection of reference genes for expression studies with fish myogenic cell cultures

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Johnston Ian A

2009-08-01

Full Text Available Abstract Background Relatively few studies have used cell culture systems to investigate gene expression and the regulation of myogenesis in fish. To produce robust data from quantitative real-time PCR mRNA levels need to be normalised using internal reference genes which have stable expression across all experimental samples. We have investigated the expression of eight candidate genes to identify suitable reference genes for use in primary myogenic cell cultures from Atlantic salmon (Salmo salar L.. The software analysis packages geNorm, Normfinder and Best keeper were used to rank genes according to their stability across 42 samples during the course of myogenic differentiation. Results Initial results showed several of the candidate genes exhibited stable expression throughout myogenic culture while Sdha was identified as the least stable gene. Further analysis with geNorm, Normfinder and Bestkeeper identified Ef1α, Hprt1, Ppia and RNApolII as stably expressed. Comparison of data normalised with the geometric average obtained from combinations of any three of these genes showed no significant differences, indicating that any combination of these genes is valid. Conclusion The geometric average of any three of Hprt1, Ef1α, Ppia and RNApolII is suitable for normalisation of gene expression data in primary myogenic cultures from Atlantic salmon.

Selection of reference genes for expression studies with fish myogenic cell cultures.

Science.gov (United States)

Bower, Neil I; Johnston, Ian A

2009-08-10

Relatively few studies have used cell culture systems to investigate gene expression and the regulation of myogenesis in fish. To produce robust data from quantitative real-time PCR mRNA levels need to be normalised using internal reference genes which have stable expression across all experimental samples. We have investigated the expression of eight candidate genes to identify suitable reference genes for use in primary myogenic cell cultures from Atlantic salmon (Salmo salar L.). The software analysis packages geNorm, Normfinder and Best keeper were used to rank genes according to their stability across 42 samples during the course of myogenic differentiation. Initial results showed several of the candidate genes exhibited stable expression throughout myogenic culture while Sdha was identified as the least stable gene. Further analysis with geNorm, Normfinder and Bestkeeper identified Ef1alpha, Hprt1, Ppia and RNApolII as stably expressed. Comparison of data normalised with the geometric average obtained from combinations of any three of these genes showed no significant differences, indicating that any combination of these genes is valid. The geometric average of any three of Hprt1, Ef1alpha, Ppia and RNApolII is suitable for normalisation of gene expression data in primary myogenic cultures from Atlantic salmon.

The Construction of cDNA Libraries from Human Single Preimplantation Embryos and Their Use in the Study of Gene Expression During Development

OpenAIRE

Adjaye, James; Daniels, Rob; Monk, Marilyn

1998-01-01

Purpose:The construction and application of polymerase chain reaction (PCR)-based cDNA libraries from unfertilized human oocytes and single preimplantation-stage embryos are described. The purpose of these studies is to provide a readily available resource for the study of gene expression during human preimplantation development.

Selection and Validation of Reference Genes for Quantitative Real-Time PCR Normalization Under Ethanol Stress Conditions in Oenococcus oeni SD-2a

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Shuai Peng

2018-05-01

Full Text Available The powerful Quantitative real-time PCR (RT-qPCR was widely used to assess gene expression levels, which requires the optimal reference genes used for normalization. Oenococcus oeni (O. oeni, as the one of most important microorganisms in wine industry and the most resistant lactic acid bacteria (LAB species to ethanol, has not been investigated regarding the selection of stable reference genes for RT-qPCR normalization under ethanol stress conditions. In this study, nine candidate reference genes (proC, dnaG, rpoA, ldhD, ddlA, rrs, gyrA, gyrB, and dpoIII were analyzed to determine the most stable reference genes for RT-qPCR in O. oeni SD-2a under different ethanol stress conditions (8, 12, and 16% (v/v ethanol. The transcript stabilities of these genes were evaluated using the algorithms geNorm, NormFinder, and BestKeeper. The results showed that dnaG and dpoIII were selected as the best reference genes across all experimental ethanol conditions. Considering single stress experimental modes, dpoIII and dnaG would be suitable to normalize expression level for 8% ethanol shock treatment, while the combination of gyrA, gyrB, and rrs would be suitable for 12% ethanol shock treatment. proC and gyrB revealed the most stable expression in 16% ethanol shock treatment. This study selected and validated for the first time the reference genes for RT-qPCR normalization in O. oeni SD-2a under ethanol stress conditions.

Real-time gene expression analysis in carp (Cyprinus carpio) skin: inflammatory responses to injury mimicking infection with ectoparasites

NARCIS (Netherlands)

Gonzalez, S.F.; Huising, M.O.; Stakauska, R.; Forlenza, M.; Verburg-van Kemenade, B.M.L.; Buchmann, K.; Nielsen, M.E.; Wiegertjes, G.F.

2007-01-01

We studied a predictive model of gene expression induced by mechanical injury of fish skin, to resolve the confounding effects on the immune system induced by injury and skin parasite-specific molecules. We applied real time quantitative PCR (RQ-PCR) to measure the expression of the pro-inflammatory

International Interlaboratory Digital PCR Study Demonstrating High Reproducibility for the Measurement of a Rare Sequence Variant.

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Whale, Alexandra S; Devonshire, Alison S; Karlin-Neumann, George; Regan, Jack; Javier, Leanne; Cowen, Simon; Fernandez-Gonzalez, Ana; Jones, Gerwyn M; Redshaw, Nicholas; Beck, Julia; Berger, Andreas W; Combaret, Valérie; Dahl Kjersgaard, Nina; Davis, Lisa; Fina, Frederic; Forshew, Tim; Fredslund Andersen, Rikke; Galbiati, Silvia; González Hernández, Álvaro; Haynes, Charles A; Janku, Filip; Lacave, Roger; Lee, Justin; Mistry, Vilas; Pender, Alexandra; Pradines, Anne; Proudhon, Charlotte; Saal, Lao H; Stieglitz, Elliot; Ulrich, Bryan; Foy, Carole A; Parkes, Helen; Tzonev, Svilen; Huggett, Jim F

2017-02-07

This study tested the claim that digital PCR (dPCR) can offer highly reproducible quantitative measurements in disparate laboratories. Twenty-one laboratories measured four blinded samples containing different quantities of a KRAS fragment encoding G12D, an important genetic marker for guiding therapy of certain cancers. This marker is challenging to quantify reproducibly using quantitative PCR (qPCR) or next generation sequencing (NGS) due to the presence of competing wild type sequences and the need for calibration. Using dPCR, 18 laboratories were able to quantify the G12D marker within 12% of each other in all samples. Three laboratories appeared to measure consistently outlying results; however, proper application of a follow-up analysis recommendation rectified their data. Our findings show that dPCR has demonstrable reproducibility across a large number of laboratories without calibration. This could enable the reproducible application of molecular stratification to guide therapy and, potentially, for molecular diagnostics.

Gene expression results in lipopolysaccharide-stimulated monocytes depend significantly on the choice of reference genes

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Øvstebø Reidun

2010-05-01

Full Text Available Abstract Background Gene expression in lipopolysaccharide (LPS-stimulated monocytes is mainly studied by quantitative real-time reverse transcription PCR (RT-qPCR using GAPDH (glyceraldehyde 3-phosphate dehydrogenase or ACTB (beta-actin as reference gene for normalization. Expression of traditional reference genes has been shown to vary substantially under certain conditions leading to invalid results. To investigate whether traditional reference genes are stably expressed in LPS-stimulated monocytes or if RT-qPCR results are dependent on the choice of reference genes, we have assessed and evaluated gene expression stability of twelve candidate reference genes in this model system. Results Twelve candidate reference genes were quantified by RT-qPCR in LPS-stimulated, human monocytes and evaluated using the programs geNorm, Normfinder and BestKeeper. geNorm ranked PPIB (cyclophilin B, B2M (beta-2-microglobulin and PPIA (cyclophilin A as the best combination for gene expression normalization in LPS-stimulated monocytes. Normfinder suggested TBP (TATA-box binding protein and B2M as the best combination. Compared to these combinations, normalization using GAPDH alone resulted in significantly higher changes of TNF-α (tumor necrosis factor-alpha and IL10 (interleukin 10 expression. Moreover, a significant difference in TNF-α expression between monocytes stimulated with equimolar concentrations of LPS from N. meningitides and E. coli, respectively, was identified when using the suggested combinations of reference genes for normalization, but stayed unrecognized when employing a single reference gene, ACTB or GAPDH. Conclusions Gene expression levels in LPS-stimulated monocytes based on RT-qPCR results differ significantly when normalized to a single gene or a combination of stably expressed reference genes. Proper evaluation of reference gene stabiliy is therefore mandatory before reporting RT-qPCR results in LPS-stimulated monocytes.

Comparison of ELISA, nested PCR and sequencing and a novel qPCR for detection of Giardia isolates from Jordan.

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Hijjawi, Nawal; Yang, Rongchang; Hatmal, Ma'mon; Yassin, Yasmeen; Mharib, Taghrid; Mukbel, Rami; Mahmoud, Sameer Alhaj; Al-Shudifat, Abdel-Ellah; Ryan, Una

2018-02-01

Little is known about the prevalence of Giardia duodenalis in human patients in Jordan and all previous studies have used direct microscopy, which lacks sensitivity. The present study developed a novel quantitative PCR (qPCR) assay at the β-giardin (bg) locus and evaluated its use as a frontline test for the diagnosis of giardiasis in comparison with a commercially available ELISA using nested PCR and sequencing of the glutamate dehydrogenase (gdh) locus (gdh nPCR) as the gold standard. A total of 96 human faecal samples were collected from 96 patients suffering from diarrhoea from 5 regions of Jordan and were screened using the ELISA and qPCR. The analytical specificity of the bg qPCR assay revealed no cross-reactions with other genera and detected all the Giardia isolates tested. Analytical sensitivity was 1 Giardia cyst per μl of DNA extract. The overall prevalence of Giardia was 64.6%. The clinical sensitivity and specificity of the bg qPCR was 89.9% and 82.9% respectively compared to 76.5 and 68.0% for the ELISA. This study is the first to compare three different methods (ELISA, bg qPCR, nested PCR and sequencing at the gdh locus) to diagnose Jordanian patients suffering from giardiasis and to analyze their demographic data. Copyright © 2018 Elsevier Inc. All rights reserved.

A comparative kinetic RT/-PCR strategy for the quantitation of mRNAs in microdissected human renal biopsy specimens.

Science.gov (United States)

Del Prete, D; Forino, M; Gambaro, G; D'Angelo, A; Baggio, B; Anglani, F

1998-01-01

Molecular biology techniques, to be applicable to a diagnostic renal biopsy specimen, should (1) be highly sensitive to be performed on a very small quantity of tissue; (2) be quantitative because they have to analyze genes normally expressed in the tissue and (3) allow the analysis of as large a number of genes as possible. Among different methods, only the reverse-transcriptase polymerase chain reaction (RT/-PCR) might comply with previous requisites, but the few RT/-PCR examples on renal biopsies in the literature do not allow starting RNA quantification and quality control; furthermore they have the drawback of analyzing only few genes. In an ongoing study to assess the expression of a number of genes in glomeruli and in tubulointerstitium of patients with different nephropathies, we developed a comparative RT/-PCR kinetic strategy based on the purification and quantification of total glomerular and tubulointerstitial RNA and on the use of an internal standard, the housekeeping gene G3PDH. We demonstrate that in microdissected diagnostic renal biopsies (1) glomerular and interstitial starting RNA can be quantified; (2) the G3PDH gene may be used both as an internal standard and as an indirect marker of RNA integrity; (3) as low as 28 ng of total RNA is sufficient to obtain PCR products of eight genes, and (4) it is worth to operate on microdissected biopsy specimens because of the different expression of genes in the two renal compartments.

Identification and validation of reference genes for qRT-PCR studies of the obligate aphid pathogenic fungus Pandora neoaphidis during different developmental stages

OpenAIRE

Zhang, Shutao; Chen, Chun; Xie, Tingna; Ye, Sudan

2017-01-01

The selection of stable reference genes is a critical step for the accurate quantification of gene expression. To identify and validate the reference genes in Pandora neoaphidis-an obligate aphid pathogenic fungus-the expression of 13classical candidate reference genes were evaluated by quantitative real-time reverse transcriptase polymerase chain reaction(qPCR) at four developmental stages (conidia, conidia with germ tubes, short hyphae and elongated hyphae). Four statistical algorithms, inc...

Amplification biases: possible differences among deviating gene expressions

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Piumi Francois

2008-01-01

Full Text Available Abstract Background Gene expression profiling has become a tool of choice to study pathological or developmental questions but in most cases the material is scarce and requires sample amplification. Two main procedures have been used: in vitro transcription (IVT and polymerase chain reaction (PCR, the former known as linear and the latter as exponential. Previous reports identified enzymatic pitfalls in PCR and IVT protocols; however the possible differences between the sequences affected by these amplification defaults were only rarely explored. Results Screening a bovine cDNA array dedicated to embryonic stages with embryonic (n = 3 and somatic tissues (n = 2, we proceeded to moderate amplifications starting from 1 μg of total RNA (global PCR or IVT one round. Whatever the tissue, 16% of the probes were involved in deviating gene expressions due to amplification defaults. These distortions were likely due to the molecular features of the affected sequences (position within a gene, GC content, hairpin number but also to the relative abundance of these transcripts within the tissues. These deviating genes mainly encoded housekeeping genes from physiological or cellular processes (70% and constituted 2 subsets which did not overlap (molecular features, signal intensities, gene ID. However, the differential expressions identified between embryonic stages were both reliable (minor intersect with biased expressions and relevant (biologically validated. In addition, the relative expression levels of those genes were biologically similar between amplified and unamplified samples. Conclusion Conversely to the most recent reports which challenged the use of intense amplification procedures on minute amounts of RNA, we chose moderate PCR and IVT amplifications for our gene profiling study. Conclusively, it appeared that systematic biases arose even with moderate amplification procedures, independently of (i the sample used: brain, ovary or embryos, (ii

The diagnosis of microorganism involved in infective endocarditis (IE by polymerase chain reaction (PCR and real-time PCR: A systematic review

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Reza Faraji

2018-02-01

Full Text Available Broad-range bacterial rDNA polymerase chain reaction (PCR followed by sequencing may be identified as the etiology of infective endocarditis (IE from surgically removed valve tissue; therefore, we reviewed the value of molecular testing in identifying organisms' DNA in the studies conducted until 2016. We searched Google Scholar, Scopus, ScienceDirect, Cochrane, PubMed, and Medline electronic databases without any time limitations up to December 2016 for English studies reporting microorganisms involved in infective endocarditis microbiology using PCR and real-time PCR. Most studies were prospective. Eleven out of 12 studies used valve tissue samples and blood cultures while only 1 study used whole blood. Also, 10 studies used the molecular method of PCR while 2 studies used real-time PCR. Most studies used 16S rDNA gene as the target gene. The bacteria were identified as the most common microorganisms involved in infective endocarditis. Streptococcus spp. and Staphylococcus spp. were, by far, the most predominant bacteria detected. In all studies, PCR and real-time PCR identified more pathogens than blood and tissue cultures; moreover, the sensitivity and specificity of PCR and real-time PCR were more than cultures in most of the studies. The highest sensitivity and specificity were 96% and 100%, respectively. The gram positive bacteria were the most frequent cause of infective endocarditis. The molecular methods enjoy a greater sensitivity compared to the conventional blood culture methods; yet, they are applicable only to the valve tissue of the patients undergoing cardiac valve surgery.

The diagnosis of microorganism involved in infective endocarditis (IE) by polymerase chain reaction (PCR) and real-time PCR: A systematic review.

Science.gov (United States)

Faraji, Reza; Behjati-Ardakani, Mostafa; Moshtaghioun, Seyed Mohammad; Kalantar, Seyed Mehdi; Namayandeh, Seyedeh Mahdieh; Soltani, Mohammadhossien; Emami, Mahmood; Zandi, Hengameh; Firoozabadi, Ali Dehghani; Kazeminasab, Mahmood; Ahmadi, Nastaran; Sarebanhassanabadi, Mohammadtaghi

2018-02-01

Broad-range bacterial rDNA polymerase chain reaction (PCR) followed by sequencing may be identified as the etiology of infective endocarditis (IE) from surgically removed valve tissue; therefore, we reviewed the value of molecular testing in identifying organisms' DNA in the studies conducted until 2016. We searched Google Scholar, Scopus, ScienceDirect, Cochrane, PubMed, and Medline electronic databases without any time limitations up to December 2016 for English studies reporting microorganisms involved in infective endocarditis microbiology using PCR and real-time PCR. Most studies were prospective. Eleven out of 12 studies used valve tissue samples and blood cultures while only 1 study used whole blood. Also, 10 studies used the molecular method of PCR while 2 studies used real-time PCR. Most studies used 16S rDNA gene as the target gene. The bacteria were identified as the most common microorganisms involved in infective endocarditis. Streptococcus spp. and Staphylococcus spp. were, by far, the most predominant bacteria detected. In all studies, PCR and real-time PCR identified more pathogens than blood and tissue cultures; moreover, the sensitivity and specificity of PCR and real-time PCR were more than cultures in most of the studies. The highest sensitivity and specificity were 96% and 100%, respectively. The gram positive bacteria were the most frequent cause of infective endocarditis. The molecular methods enjoy a greater sensitivity compared to the conventional blood culture methods; yet, they are applicable only to the valve tissue of the patients undergoing cardiac valve surgery. Copyright © 2017. Published by Elsevier Taiwan.

A study on the effect of Helicobacter pylori infection on p53 expression in gastric cancer and gastritis tissues.

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Salih, Barik A; Gucin, Zuhal; Bayyurt, Nizamettin

2013-09-16

Helicobacter pylori cause damage to gastric epithelial cells and alterations in the p53 gene that lead to cancer development. This study aimed to determine the correlation of p53 expression with H. pylori using immunohistochemistry, RFLP-PCR, and histopathology. Gastric biopsy samples from gastric cancer (GC) (n = 54) and gastritis (n = 31) patients were examined for histopathological changes and expression of p53 protein by immunohistochemistry. Immunohistochemical analysis of p53 protein expression in H. pylori-positive GC sections showed an average of 44.3% positive cells in tumors and 6.9% in normal tissues, as compared to 16.4% and 4.4% in H. pylori-negative sections. P53 expression showed significant association with H. pylori (P = 0.005), invasion depth (P = 0.029) and inflammation reaction (P = 0.008). In gastritis sections, no difference in the average p53 staining in H. pylori-positive or -negative sections was seen. PCR-RFLP results also showed no difference in genotype frequencies of p53 in H. pylori-positive or -negative gastritis sections. Histopathology study of H. pylori-positive GC sections showed that 97.2% were the intestinal type and 2.8% the diffuse type, while in H. pylori-negative sections 35.2% were the intestinal type and 64.8% the diffuse type. Biopsy sections from H. pylori-positive gastritis patients revealed more severe inflammation than those of H. pylori-negative patients. Our results show that H. pylori infection affects p53 expression in GC. The average p53 expression was significantly higher in tumor than in normal tissues. In gastritis sections p53 expression was significantly associated with H. pylori.

Molecular characterization and expression of maternally expressed gene 3 (Meg3/Gtl2) RNA in the mouse inner ear

DEFF Research Database (Denmark)

Manji, S.S.; Sørensen, Brita Singers; Klockars, T.

2006-01-01

The pathways responsible for sound perception in the cochlea involve the coordinated and regulated expression of hundreds of genes. By using microarray analysis, we identified several transcripts enriched in the inner ear, including the maternally expressed gene 3 (Meg3/Gtl2), an imprinted...... noncoding RNA. Real-time PCR analysis demonstrated that Meg3/Gtl2 was highly expressed in the cochlea, brain, and eye. Molecular studies revealed the presence of several Meg3/Gtl2 RNA splice variants in the mouse cochlea, brain, and eye. In situ hybridizations showed intense Meg3/Gtl2 RNA staining...... otocyst and localized to the spiral ganglion, stria vascularis, Reissner's membrane, and greater epithelial ridge (GER) in the cochlear duct. RT-PCR analysis performed on cell lines derived from the organ of Corti, representing neural, supporting, and hair cells, showed significantly elevated levels...

Data on individual PCR efficiency values as quality control for circulating miRNAs

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Anna Brunet-Vega

2015-12-01

Full Text Available This data article contains data related to the research article entitled “Variability in microRNA recovery from plasma: Comparison of five commercial kits, doi:10.1016/j.ab.2015.07.018” Brunet-Vega (2015 [1]. PCR efficiency, along with RNA and cDNA quality, are the most important factors affecting the quality of qPCR results. Constant amplification efficiency in all compared samples is indispensable when relative quantification is used to measure changes in gene expression. An easy way to measure PCR efficiency, without the need of a standard curve, is LinRegPCR software. Individual PCR efficiency can be determined as a part of qPCR quality control. This is especially important when the initial RNA quantity is so low that cannot be accurately quantified, such as in circulating RNA extractions. This data article reports the Cqs and PCR efficiencies of 5 miRNAs quantified in RNA isolated from 4 patients with colorectal cancer (CRC and 4 healthy donors using five commercially available kits.

Material Biocompatibility for PCR Microfluidic Chips

KAUST Repository

Kodzius, Rimantas

2010-04-23

As part of the current miniaturization trend, biological reactions and processes are being adapted to microfluidics devices. PCR is the primary method employed in DNA amplification, its miniaturization is central to efforts to develop portable devices for diagnostics and testing purposes. A problem is the PCR-inhibitory effect due to interaction between PCR reagents and the surrounding environment, which effect is increased in high-surface-are-to-volume ration microfluidics. In this study, we evaluated the biocompatibility of various common materials employed in the fabrication of microfluidic chips, including silicon, several kinds of silicon oxide, glasses, plastics, wax, and adhesives. Two-temperature PCR was performed with these materials to determine their PCR-inhibitory effect. In most of the cases, addition of bovine serum albumin effectively improved the reaction yield. We also studied the individual PCR components from the standpoint of adsorption. Most of the materials did not inhibit the DNA, whereas they did show noticeable interaction with the DNA polymerase. Our test, instead of using microfluidic devices, can be easily conducted in common PCR tubes using a standard bench thermocycler. Our data supports an overview of the means by which the materials most bio-friendly to microfluidics can be selected.

Material Biocompatibility for PCR Microfluidic Chips

KAUST Repository

Kodzius, Rimantas; Chang, Donald Choy; Gong, Xiuqing; Wen, Weijia; Wu, Jinbo; Xiao, Kang; Yi, Xin

2010-01-01

As part of the current miniaturization trend, biological reactions and processes are being adapted to microfluidics devices. PCR is the primary method employed in DNA amplification, its miniaturization is central to efforts to develop portable devices for diagnostics and testing purposes. A problem is the PCR-inhibitory effect due to interaction between PCR reagents and the surrounding environment, which effect is increased in high-surface-are-to-volume ration microfluidics. In this study, we evaluated the biocompatibility of various common materials employed in the fabrication of microfluidic chips, including silicon, several kinds of silicon oxide, glasses, plastics, wax, and adhesives. Two-temperature PCR was performed with these materials to determine their PCR-inhibitory effect. In most of the cases, addition of bovine serum albumin effectively improved the reaction yield. We also studied the individual PCR components from the standpoint of adsorption. Most of the materials did not inhibit the DNA, whereas they did show noticeable interaction with the DNA polymerase. Our test, instead of using microfluidic devices, can be easily conducted in common PCR tubes using a standard bench thermocycler. Our data supports an overview of the means by which the materials most bio-friendly to microfluidics can be selected.

MAEWEST expression in flower development of two petunia species.

Science.gov (United States)

Segatto, Ana Lúcia A; Turchetto-Zolet, Andreia Carina; Aizza, Lilian Cristina B; Monte-Bello, Carolina C; Dornelas, Marcelo C; Margis, Rogerio; Freitas, Loreta B

2013-07-03

Changes in flower morphology may influence the frequency and specificity of animal visitors. In Petunia (Solanaceae), adaptation to different pollinators is one of the factors leading to species diversification within the genus. This study provides evidence that differential expression patterns of MAWEWEST (MAW) homologs in different Petunia species may be associated with adaptive changes in floral morphology. The Petunia × hybrida MAW gene belongs to the WOX (WUSCHEL-related homeobox) transcription factor family and has been identified as a controller of petal fusion during corolla formation. We analyzed the expression patterns of P. inflata and P. axillaris MAW orthologs (PiMAW and PaMAW, respectively) by reverse transcriptase polymerase chain reaction (RT-PCR), reverse transcription-quantitative PCR (qRT-PCR) and in situ hybridization in different tissues and different developmental stages of flowers in both species. The spatial expression patterns of PiMAW and PaMAW were similar in P. inflata and P. axillaris. Nevertheless, PaMAW expression level in P. axillaris was higher during the late bud development stage as compared to PiMAW in P. inflata. This work represents an expansion of petunia developmental research to wild accessions.

MAEWEST Expression in Flower Development of Two Petunia Species

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Ana Lúcia A. Segatto

2013-07-01

Full Text Available Changes in flower morphology may influence the frequency and specificity of animal visitors. In Petunia (Solanaceae, adaptation to different pollinators is one of the factors leading to species diversification within the genus. This study provides evidence that differential expression patterns of MAWEWEST (MAW homologs in different Petunia species may be associated with adaptive changes in floral morphology. The Petunia × hybrida MAW gene belongs to the WOX (WUSCHEL-related homeobox transcription factor family and has been identified as a controller of petal fusion during corolla formation. We analyzed the expression patterns of P. inflata and P. axillaris MAW orthologs (PiMAW and PaMAW, respectively by reverse transcriptase polymerase chain reaction (RT-PCR, reverse transcription–quantitative PCR (qRT-PCR and in situ hybridization in different tissues and different developmental stages of flowers in both species. The spatial expression patterns of PiMAW and PaMAW were similar in P. inflata and P. axillaris. Nevertheless, PaMAW expression level in P. axillaris was higher during the late bud development stage as compared to PiMAW in P. inflata. This work represents an expansion of petunia developmental research to wild accessions.

OCT-4 expression in follicular and luteal phase endometrium: a pilot study

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Huber Johannes C

2010-04-01

Full Text Available Abstract Background The stem cell marker Octamer-4 (OCT-4 is expressed in human endometrium. Menstrual cycle-dependency of OCT-4 expression has not been investigated to date. Methods In a prospective, single center cohort study of 98 women undergoing hysteroscopy during the follicular (n = 49 and the luteal (n = 40 phases of the menstrual cycle, we obtained endometrial samples. Specimens were investigated for OCT-4 expression on the mRNA and protein levels using reverse transcriptase polymerase chain reaction (RT-PCR and immunohistochemistry. Expression of OCT-4 was correlated to menstrual cycle phase. Results Of 89 women sampled, 49 were in the follicular phase and 40 were in the luteal phase. OCT-4 mRNA was detected in all samples. Increased OCT-4 mRNA levels in the follicular and luteal phases was found in 35/49 (71% and 27/40 (68% of women, respectively (p = 0.9. Increased expression of OCT-4 protein was identified in 56/89 (63% samples. Increased expression of OCT-4 protein in the follicular and luteal phases was found in 33/49 (67% and 23/40 (58% of women, respectively (p = 0.5. Conclusions On the mRNA and protein levels, OCT-4 is not differentially expressed during the menstrual cycle. Endometrial OCT-4 is not involved in or modulated by hormone-induced cyclical changes of the endometrium.

Study comparing human papillomavirus (HPV) real-time multiplex PCR and Hybrid Capture II INNO-LiPA v2 HPV genotyping PCR assays

DEFF Research Database (Denmark)

Iftner, Thomas; Germ, Liesje; Swoyer, Ryan

2009-01-01

methods has not been well characterized. Clinically, cytology is used to establish possible HPV infection. We evaluated the sensitivity and specificity of HPV multiplex PCR assays compared to those of the testing scheme of the Hybrid Capture II (HCII) assay followed by an HPV PCR/line hybridization assay...... (HCII-LiPA v2). SurePath residual samples were split into two aliquots. One aliquot was subjected to HCII testing followed by DNA extraction and LiPA v2 genotyping. The second aliquot was shipped to a second laboratory, where DNA was extracted and HPV multiplex PCR testing was performed. Comparisons...... were evaluated for 15 HPV types common in both assays. A slightly higher proportion of samples tested positive by the HPV multiplex PCR than by the HCII-LiPA v2 assay. The sensitivities of the multiplex PCR assay relative to those of the HCII-LiPA v2 assay for HPV types 6, 11, 16, and 18, for example...

[E-MTAB-587] PCR_artifacts

NARCIS (Netherlands)

Muino Acuna, J.M.

2011-01-01

WARNING: This library was yield low amount of material and it was over-amplified by PCR. This libraries are used study the robustness of several statitical methods against PCR artifacts. ChIP experiments were performed on Arabidopsis wildtype inflorescences using an antibody raised against a

Normalization with Corresponding Naïve Tissue Minimizes Bias Caused by Commercial Reverse Transcription Kits on Quantitative Real-Time PCR Results.

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Andreas Garcia-Bardon

Full Text Available Real-time reverse transcription polymerase chain reaction (PCR is the gold standard for expression analysis. Designed to improve reproducibility and sensitivity, commercial kits are commonly used for the critical step of cDNA synthesis. The present study was designed to determine the impact of these kits. mRNA from mouse brains were pooled to create serial dilutions ranging from 0.0625 μg to 2 μg, which were transcribed into cDNA using four different commercial reverse-transcription kits. Next, we transcribed mRNA from brain tissue after acute brain injury and naïve mice into cDNA for qPCR. Depending on tested genes, some kits failed to show linear results in dilution series and revealed strong variations in cDNA yield. Absolute expression data in naïve and trauma settings varied substantially between these kits. Normalization with a housekeeping gene failed to reduce kit-dependent variations, whereas normalization eliminated differences when naïve samples from the same region were used. The study shows strong evidence that choice of commercial cDNA synthesis kit has a major impact on PCR results and, consequently, on comparability between studies. Additionally, it provides a solution to overcome this limitation by normalization with data from naïve samples. This simple step helps to compare mRNA expression data between different studies and groups.

Real-time gene expression analysis in carp (Cyprinus carpio L.) skin: Inflammatory responses to injury mimicking infection with ectoparasites

NARCIS (Netherlands)

Gonzalez, S.F.; Huising, M.O.; Stakauskas, R.; Forlenza, M.; Verburg-van Kemenade, B.M.L.; Buchmann, K.; Nielsen, M.E.; Wiegertjes, G.F.

2007-01-01

We studied a predictive model of gene expression induced by mechanical injury of fish skin, to resolve the confounding effects on the immune system induced by injury and skin parasite-specific molecules. We applied real time quantitative PCR (RQ-PCR) to measure the expression of the pro-inflammatory

Selection of suitable housekeeping genes for real-time quantitative PCR in CD4(+ lymphocytes from asthmatics with or without depression.

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Ting Wang

Full Text Available OBJECTIVE: No optimal housekeeping genes (HKGs have been identified for CD4(+ T cells from non-depressive asthmatic and depressive asthmatic adults for normalizing quantitative real-time PCR (qPCR assays. The aim of present study was to select appropriate HKGs for gene expression analysis in purified CD4(+ T cells from these asthmatics. METHODS: Three groups of subjects (Non-depressive asthmatic, NDA, n = 10, Depressive asthmatic, DA, n = 11, and Healthy control, HC, n = 10 respectively were studied. qPCR for 9 potential HKGs, namely RNA, 28S ribosomal 1 (RN28S1, ribosomal protein, large, P0 (RPLP0, actin, beta (ACTB, cyclophilin A (PPIA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, phosphoglycerate kinase 1 (PGK1, beta-2-microglobulin (B2M, glucuronidase, beta (GUSB and ribosomal protein L13a (RPL13A, was performed. Then the data were analyzed with three different applications namely BestKeeper, geNorm, and NormFinder. RESULTS: The analysis of gene expression data identified B2M and RPLP0 as the most stable reference genes and showed that the level of PPIA was significantly different among subjects of three groups when the two best HKGs identified were applied. Post-hoc analysis by Student-Newman-Keuls correction shows that depressive asthmatics and non-depressive asthmatics exhibited lower expression level of PPIA than healthy controls (p<0.05. CONCLUSIONS: B2M and RPLP0 were identified as the most optimal HKGs in gene expression studies involving human blood CD4(+ T cells derived from normal, depressive asthmatics and non-depressive asthmatics. The suitability of using the PPIA gene as the HKG for such studies was questioned due to its low expression in asthmatics.

Identification of appropriate reference genes for human mesenchymal stem cell analysis by quantitative real-time PCR.

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Li, Xiuying; Yang, Qiwei; Bai, Jinping; Xuan, Yali; Wang, Yimin

2015-01-01

Normalization to a reference gene is the method of choice for quantitative reverse transcription-PCR (RT-qPCR) analysis. The stability of reference genes is critical for accurate experimental results and conclusions. We have evaluated the expression stability of eight commonly used reference genes found in four different human mesenchymal stem cells (MSC). Using geNorm, NormFinder and BestKeeper algorithms, we show that beta-2-microglobulin and peptidyl-prolylisomerase A were the optimal reference genes for normalizing RT-qPCR data obtained from MSC, whereas the TATA box binding protein was not suitable due to its extensive variability in expression. Our findings emphasize the significance of validating reference genes for qPCR analyses. We offer a short list of reference genes to use for normalization and recommend some commercially-available software programs as a rapid approach to validate reference genes. We also demonstrate that the two reference genes, β-actin and glyceraldehyde-3-phosphate dehydrogenase, are frequently used are not always successful in many cases.

Differential expression of granulopoiesis related genes in neutrophil subsets distinguished by membrane expression of CD177

DEFF Research Database (Denmark)

Hu, Nan; Mora-Jensen, Helena; Theilgaard-Mønch, Kim

2014-01-01

OBJECTIVE: Differential gene expression in CD177+ and CD177- neutrophils was investigated, in order to detect possible differences in neutrophil function which could be related to the pathogenesis of ANCA-associated Vasculitides (AAV). METHODS: Neutrophils were isolated from healthy controls (HC......) with high, negative or bimodal CD177 expression, and sorted into CD177+ and CD177- subpopulations. Total RNA was screened for expression of 24,000 probes with Illumina Ref-8 Beadchips. Genes showing differential expression between CD177+ and CD177- subsets in microarray analysis were re-assessed using...... quantitative-PCR. CD177 expression on neutrophil precursors in bone marrow was analyzed using quantitative PCR and flowcytometry. RESULTS: The proportion of CD177+ cells increased during neutrophil maturation in bone marrow. Fold change analysis of gene expression profile of sorted CD177+ and CD177...

Sleep Deprivation Influences Circadian Gene Expression in the Lateral Habenula.

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Zhang, Beilin; Gao, Yanxia; Li, Yang; Yang, Jing; Zhao, Hua

2016-01-01

Sleep is governed by homeostasis and the circadian clock. Clock genes play an important role in the generation and maintenance of circadian rhythms but are also involved in regulating sleep homeostasis. The lateral habenular nucleus (LHb) has been implicated in sleep-wake regulation, since LHb gene expression demonstrates circadian oscillation characteristics. This study focuses on the participation of LHb clock genes in regulating sleep homeostasis, as the nature of their involvement is unclear. In this study, we observed changes in sleep pattern following sleep deprivation in LHb-lesioned rats using EEG recording techniques. And then the changes of clock gene expression (Per1, Per2, and Bmal1) in the LHb after 6 hours of sleep deprivation were detected by using real-time quantitative PCR (qPCR). We found that sleep deprivation increased the length of Non-Rapid Eye Movement Sleep (NREMS) and decreased wakefulness. LHb-lesioning decreased the amplitude of reduced wake time and increased NREMS following sleep deprivation in rats. qPCR results demonstrated that Per2 expression was elevated after sleep deprivation, while the other two genes were unaffected. Following sleep recovery, Per2 expression was comparable to the control group. This study provides the basis for further research on the role of LHb Per2 gene in the regulation of sleep homeostasis.

Stem-Loop RT-qPCR as an Efficient Tool for the Detection and Quantification of Small RNAs in Giardia lamblia

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Jaime Marcial-Quino

2016-12-01

Full Text Available Stem-loop quantitative reverse transcription PCR (RT-qPCR is a molecular technique used for identification and quantification of individual small RNAs in cells. In this work, we used a Universal ProbeLibrary (UPL-based design to detect—in a rapid, sensitive, specific, and reproducible way—the small nucleolar RNA (snoRNA GlsR17 and its derived miRNA (miR2 of Giardia lamblia using a stem-loop RT-qPCR approach. Both small RNAs could be isolated from both total RNA and small RNA samples. Identification of the two small RNAs was carried out by sequencing the PCR-amplified small RNA products upon ligation into the pJET1.2/blunt vector. GlsR17 is constitutively expressed during the 72 h cultures of trophozoites, while the mature miR2 is present in 2-fold higher abundance during the first 48 h than at 72 h. Because it has been suggested that miRNAs in G. lamblia have an important role in the regulation of gene expression, the use of the stem-loop RT-qPCR method could be valuable for the study of miRNAs of G. lamblia. This methodology will be a powerful tool for studying gene regulation in G. lamblia, and will help to better understand the features and functions of these regulatory molecules and how they work within the RNA interference (RNAi pathway in G. lamblia.

Stem-Loop RT-qPCR as an Efficient Tool for the Detection and Quantification of Small RNAs in Giardia lamblia

Science.gov (United States)

Marcial-Quino, Jaime; Gómez-Manzo, Saúl; Fierro, Francisco; Vanoye-Carlo, America; Rufino-González, Yadira; Sierra-Palacios, Edgar; Castillo-Villanueva, Adriana; Castillo-Rodríguez, Rosa Angélica; Rodríguez-Bustamante, Eduardo; Arreguin-Espinosa, Roberto; Reyes-Vivas, Horacio

2016-01-01

Stem-loop quantitative reverse transcription PCR (RT-qPCR) is a molecular technique used for identification and quantification of individual small RNAs in cells. In this work, we used a Universal ProbeLibrary (UPL)-based design to detect—in a rapid, sensitive, specific, and reproducible way—the small nucleolar RNA (snoRNA) GlsR17 and its derived miRNA (miR2) of Giardia lamblia using a stem-loop RT-qPCR approach. Both small RNAs could be isolated from both total RNA and small RNA samples. Identification of the two small RNAs was carried out by sequencing the PCR-amplified small RNA products upon ligation into the pJET1.2/blunt vector. GlsR17 is constitutively expressed during the 72 h cultures of trophozoites, while the mature miR2 is present in 2-fold higher abundance during the first 48 h than at 72 h. Because it has been suggested that miRNAs in G. lamblia have an important role in the regulation of gene expression, the use of the stem-loop RT-qPCR method could be valuable for the study of miRNAs of G. lamblia. This methodology will be a powerful tool for studying gene regulation in G. lamblia, and will help to better understand the features and functions of these regulatory molecules and how they work within the RNA interference (RNAi) pathway in G. lamblia. PMID:27999395

Comparison of kDNA PCR-hybridization assay with three PCR methods for canines visceral Leishmaniasis diagnosis

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Pilatti, Marcia M.; Andrade, Antero S.R. [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil)], e-mail: marciapilatti@yahoo.com.br, e-mail: antero@cdtn.br; Ferreira, Sidney A. [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Dept. de Parasitologia], e-mail: saninoalmeida@gmail.com

2009-07-01

The sensitivity of the kDNA PCR-Hybridization assay, which uses radioactive DNA probes (labeled with {sup 32}P), was compared with three conventional PCR methods used for canine visceral leishmaniasis diagnosis. All PCR methods had two steps: a first amplification followed by hybridization or by a new amplification (nested or semi nested). Two methods (kDNA PCR-Hybridization and kDNA snPCR) used primers addressed to kinetoplast minicircles and the other two methods to the coding (LnPCR) and intergenic noncoding regions (ITS-1 nPCR) of the ribosomal rRNA genes. The comparison was accomplished in two groups of 23 infected dogs using samples collected by the conjunctival swab procedure. In the Group 1 the DNA was extracted from cotton swabs by phenol-chloroform and in Group 2 by boiling. The most efficient PCR methods in the Group 1 were those based on kDNA targets. The kDNA PCR-Hybridization was able to detect parasites in 22/23 dogs (95.6%) and in 40/46 samples (86.9%). The kDNA snPCR was positive for 21/23 dogs (91.3%) and for 40/46 samples (86.9%). The positivities of the kDNA based methods were significantly higher than the positivities verified for the methods based on ribosomal rRNA genes (p<0.05). In the Group 2 the kDNA PCR- Hybridization showed a better performance detecting parasites in 18/23 dogs (78.3%) and in 31/46 samples (67.4%), significantly higher than the other three methods (p<0.05). The higher sensitivity of the minicircle kDNA based assays reported by others was confirmed in this study and kDNA PCR-Hybridization showed the best sensitivity among the assays evaluated. (author)

Comparison of kDNA PCR-hybridization assay with three PCR methods for canines visceral Leishmaniasis diagnosis

International Nuclear Information System (INIS)

Pilatti, Marcia M.; Andrade, Antero S.R.; Ferreira, Sidney A.

2009-01-01

The sensitivity of the kDNA PCR-Hybridization assay, which uses radioactive DNA probes (labeled with 32 P), was compared with three conventional PCR methods used for canine visceral leishmaniasis diagnosis. All PCR methods had two steps: a first amplification followed by hybridization or by a new amplification (nested or semi nested). Two methods (kDNA PCR-Hybridization and kDNA snPCR) used primers addressed to kinetoplast minicircles and the other two methods to the coding (LnPCR) and intergenic noncoding regions (ITS-1 nPCR) of the ribosomal rRNA genes. The comparison was accomplished in two groups of 23 infected dogs using samples collected by the conjunctival swab procedure. In the Group 1 the DNA was extracted from cotton swabs by phenol-chloroform and in Group 2 by boiling. The most efficient PCR methods in the Group 1 were those based on kDNA targets. The kDNA PCR-Hybridization was able to detect parasites in 22/23 dogs (95.6%) and in 40/46 samples (86.9%). The kDNA snPCR was positive for 21/23 dogs (91.3%) and for 40/46 samples (86.9%). The positivities of the kDNA based methods were significantly higher than the positivities verified for the methods based on ribosomal rRNA genes (p<0.05). In the Group 2 the kDNA PCR- Hybridization showed a better performance detecting parasites in 18/23 dogs (78.3%) and in 31/46 samples (67.4%), significantly higher than the other three methods (p<0.05). The higher sensitivity of the minicircle kDNA based assays reported by others was confirmed in this study and kDNA PCR-Hybridization showed the best sensitivity among the assays evaluated. (author)

PCR-SSCP analysis and its application to human genome study

International Nuclear Information System (INIS)

Hayashi, Kenshi

1994-01-01

A large amount of DNA sequence data are now available owing to the development of the human genome project. These data are deposited in public databases, e.g. DDBJ, GebBank and EMBL, and freely accessible to scientific community. One of the major advantages of having these databases is that we can now detect sequence differences between individuals in a large scale. Using the sequence informations, we can design primer sequences, amplify various target regions of the sample DNA's by PCR and detect abnormal sequence changes from reference, or normal sequences. Detecting sequence changes, or mutations, are essential part of searching genes responsible for hereditary diseases and also DNA diagnosis of hereditary diseases or cancer. We can also measure mutation frequency of the human genome by knowing its variability. Our group has developed and been improving a method, PCR-SSCP analysis, as an extremely rapid and easy technique for detection of sequence differences between sample DNA's. Knowing the sensitivity (percentage detection of mutations) of this technique is important in evaluating usefulness of it for the purposes stated above. Considerable number of experiences on PCR-SSCP analysis of fragments shorter than 300 b.p. are accumulating. We summarize here the sensitivity of PCR-SSCP analysis for various sequence context of this size range examined in various electrophoretic conditions conducted in many laboratories. Data on mutation detection by this technique for longer fragments are limited. We also present oue effort for defining electrophoretic conditions of PCR-SSCP analysis when examining longer (350 to 600 b.p.) fragments. (author)

MicroRNA Expression Varies according to Glucose Tolerance, Measurement Platform, and Biological Source

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S. Dias

2017-01-01

Full Text Available Dysregulated microRNA (miRNA expression is observed during type 2 diabetes (T2D, although the consistency of miRNA expression across measurement platform and biological source is uncertain. Here we report miRNA profiling in the whole blood and serum of South African women with different levels of glucose tolerance, using next generation sequencing (NGS and quantitative real time PCR (qRT-PCR. Whole blood-derived miRNAs from women with newly diagnosed T2D (n=4, impaired glucose tolerance (IGT (n=4, and normal glucose tolerance (NGT (n=4 were subjected to NGS, whereafter transcript levels of selected miRNAs were quantified in the whole blood and serum of these women using qRT-PCR. Of the five significantly differentially expressed miRNAs identified by NGS, only the directional increase of miR-27b in women with IGT compared to NGT was confirmed in whole blood and serum, using qRT-PCR. Functional enrichment of miR-27b gene targets identified biological pathways associated with glucose transport and insulin regulation. In conclusion, this study showed poor correlation in miRNA expression profiled using NGS and qRT-PCR and in whole blood and serum. The consistent increased expression of miR-27b in women with IGT compared to NGT across measurement platform and biological source holds potential as a biomarker for risk stratification in our population.

Study of the Efficacy of Real Time-PCR Method for Amikacin Determination Using Microbial Assay

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Farzaneh Lotfipour

2015-06-01

Full Text Available Purpose: Microbial assay is used to determine the potency of antibiotics and vitamins. In spite of its advantages like simplicity and easiness, and to reveal the slight changes in the molecules, the microbial assay suffers from significant limitations; these methods are of lower specificity, accuracy and sensitivity. The objective of the present study is to evaluate the efficacy of real time-PCR technique in comparison with turbidimetric method for microbial assay of amikacin. Methods: Microbial determination of amikacin by turbidimetric method was performed according to USP. Also amikacin concentrations were determined by microbial assay using taq-man quantitative PCR method. Standard curves in different concentration for both methods were plotted and method validation parameters of linearity, precision and accuracy were calculated using statistical procedures. Results: The RT-PCR method was linear in the wider concentration range (5.12 – 38.08 for RT-PCR versus 8.00 – 30.47 for turbidimetric method with a better correlation coefficient (0.976 for RT-PCR versus 0.958 for turbidimetric method. RT-PCR method with LOQ of 5.12 ng/ml was more sensitive than turbidimetric method with LOQ of 8.00 ng/ml and the former could detect and quantify low concentrations of amikacin. The results of accuracy and precision evaluation showed that the RT-PCR method was accurate and precise in all of the tested concentration. Conclusion: The RT-PCR method described here provided an accurate and precise technique for measurement of amikacin potency and it can be a candidate for microbial determination of the antibiotics with the same test organism.

Reference gene selection for qRT-PCR assays in Stellera chamaejasme subjected to abiotic stresses and hormone treatments based on transcriptome datasets.

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Liu, Xin; Guan, Huirui; Song, Min; Fu, Yanping; Han, Xiaomin; Lei, Meng; Ren, Jingyu; Guo, Bin; He, Wei; Wei, Yahui

2018-01-01

Stellera chamaejasme Linn, an important poisonous plant of the China grassland, is toxic to humans and livestock. The rapid expansion of S. chamaejasme has greatly damaged the grassland ecology and, consequently, seriously endangered the development of animal husbandry. To draft efficient prevention and control measures, it has become more urgent to carry out research on its adaptive and expansion mechanisms in different unfavorable habitats at the genetic level. Quantitative real-time polymerase chain reaction (qRT-PCR) is a widely used technique for studying gene expression at the transcript level; however, qRT-PCR requires reference genes (RGs) as endogenous controls for data normalization and only through appropriate RG selection and qRT-PCR can we guarantee the reliability and robustness of expression studies and RNA-seq data analysis. Unfortunately, little research on the selection of RGs for gene expression data normalization in S. chamaejasme has been reported. In this study, 10 candidate RGs namely, 18S , 60S , CYP , GAPCP1 , GAPDH2 , EF1B , MDH , SAND , TUA1 , and TUA6 , were singled out from the transcriptome database of S. chamaejasme , and their expression stability under three abiotic stresses (drought, cold, and salt) and three hormone treatments (abscisic acid, ABA; gibberellin, GA; ethephon, ETH) were estimated with the programs geNorm, NormFinder, and BestKeeper. Our results showed that GAPCP1 and EF1B were the best combination for the three abiotic stresses, whereas TUA6 and SAND , TUA1 and CYP , GAPDH2 and 60S were the best choices for ABA, GA, and ETH treatment, respectively. Moreover, GAPCP1 and 60S were assessed to be the best combination for all samples, and 18S was the least stable RG for use as an internal control in all of the experimental subsets. The expression patterns of two target genes ( P5CS2 and GI ) further verified that the RGs that we selected were suitable for gene expression normalization. This work is the first attempt to

Subtractive hybridization and random arbitrarily primed PCR analyses of a benzoate-assimilating bacterium, Desulfotignum balticum.

Science.gov (United States)

Habe, Hiroshi; Kobuna, Akinori; Hosoda, Akifumi; Kouzuma, Atsushi; Yamane, Hisakazu; Nojiri, Hideaki; Omori, Toshio; Watanabe, Kazuya

2008-05-01

Subtractive hybridization (SH) and random arbitrarily primed PCR (RAP-PCR) were used to detect genes involved in anaerobic benzoate degradation by Desulfotignum balticum. Through SH, we obtained 121 DNA sequences specific for D. balticum but not for D. phosphitoxidans (a non-benzoate-assimilating species). Furthermore, RAP-PCR analysis showed that a 651-bp DNA fragment, having 55% homology with the solute-binding protein of the ABC transporter system in Methanosarcina barkeri, was expressed when D. balticum was grown on benzoate, but not on pyruvate. By shotgun sequencing of the fosmid clone (38,071 bp) containing the DNA fragment, 33 open reading frames (ORFs) and two incomplete ORFs were annotated, and several genes within this region corresponded to the DNA fragments obtained by SH. An 11.3-kb gene cluster (ORF10-17) revealed through reverse transcription-PCR showed homology with the ABC transporter system and TonB-dependent receptors, both of which are presumably involved in the uptake of siderophore/heme/vitamin B(12), and was expressed in response to growth on benzoate.

Accurate quantification of supercoiled DNA by digital PCR

Science.gov (United States)

Dong, Lianhua; Yoo, Hee-Bong; Wang, Jing; Park, Sang-Ryoul

2016-01-01

Digital PCR (dPCR) as an enumeration-based quantification method is capable of quantifying the DNA copy number without the help of standards. However, it can generate false results when the PCR conditions are not optimized. A recent international comparison (CCQM P154) showed that most laboratories significantly underestimated the concentration of supercoiled plasmid DNA by dPCR. Mostly, supercoiled DNAs are linearized before dPCR to avoid such underestimations. The present study was conducted to overcome this problem. In the bilateral comparison, the National Institute of Metrology, China (NIM) optimized and applied dPCR for supercoiled DNA determination, whereas Korea Research Institute of Standards and Science (KRISS) prepared the unknown samples and quantified them by flow cytometry. In this study, several factors like selection of the PCR master mix, the fluorescent label, and the position of the primers were evaluated for quantifying supercoiled DNA by dPCR. This work confirmed that a 16S PCR master mix avoided poor amplification of the supercoiled DNA, whereas HEX labels on dPCR probe resulted in robust amplification curves. Optimizing the dPCR assay based on these two observations resulted in accurate quantification of supercoiled DNA without preanalytical linearization. This result was validated in close agreement (101~113%) with the result from flow cytometry. PMID:27063649

Temperature and Development Impacts on Housekeeping Gene Expression in Cowpea Aphid, Aphis craccivora (Hemiptera: Aphidiae.

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Chunxiao Yang

Full Text Available Quantitative real-time PCR (qRT-PCR is a powerful technique to quantify gene expression. To standardize gene expression studies and obtain more accurate qRT-PCR analysis, normalization relative to consistently expressed housekeeping genes (HKGs is required. In this study, ten candidate HKGs including elongation factor 1 α (EF1A, ribosomal protein L11 (RPL11, ribosomal protein L14 (RPL14, ribosomal protein S8 (RPS8, ribosomal protein S23 (RPS23, NADH-ubiquinone oxidoreductase (NADH, vacuolar-type H+-ATPase (ATPase, heat shock protein 70 (HSP70, 18S ribosomal RNA (18S, and 12S ribosomal RNA (12S from the cowpea aphid, Aphis craccivora Koch were selected. Four algorithms, geNorm, Normfinder, BestKeeper, and the ΔCt method were employed to evaluate the expression profiles of these HKGs as endogenous controls across different developmental stages and temperature regimes. Based on RefFinder, which integrates all four analytical algorithms to compare and rank the candidate HKGs, RPS8, RPL14, and RPL11 were the three most stable HKGs across different developmental stages and temperature conditions. This study is the first step to establish a standardized qRT-PCR analysis in A. craccivora following the MIQE guideline. Results from this study lay a foundation for the genomics and functional genomics research in this sap-sucking insect pest with substantial economic impact.

Selection of relatively exact reference genes for gene expression studies in goosegrass (Eleusine indica) under herbicide stress.

Science.gov (United States)

Chen, Jingchao; Huang, Zhaofeng; Huang, Hongjuan; Wei, Shouhui; Liu, Yan; Jiang, Cuilan; Zhang, Jie; Zhang, Chaoxian

2017-04-21

Goosegrass (Eleusine indica) is one of the most serious annual grassy weeds worldwide, and its evolved herbicide-resistant populations are more difficult to control. Quantitative real-time PCR (qPCR) is a common technique for investigating the resistance mechanism; however, there is as yet no report on the systematic selection of stable reference genes for goosegrass. This study proposed to test the expression stability of 9 candidate reference genes in goosegrass in different tissues and developmental stages and under stress from three types of herbicide. The results show that for different developmental stages and organs (control), eukaryotic initiation factor 4 A (eIF-4) is the most stable reference gene. Chloroplast acetolactate synthase (ALS) is the most stable reference gene under glyphosate stress. Under glufosinate stress, eIF-4 is the best reference gene. Ubiquitin-conjugating enzyme (UCE) is the most stable reference gene under quizalofop-p-ethyl stress. The gene eIF-4 is the recommended reference gene for goosegrass under the stress of all three herbicides. Moreover, pairwise analysis showed that seven reference genes were sufficient to normalize the gene expression data under three herbicides treatment. This study provides a list of reliable reference genes for transcript normalization in goosegrass, which will facilitate resistance mechanism studies in this weed species.

Systematic analysis of gene expression pattern in has-miR-197 over-expressed human uterine leiomyoma cells.

Science.gov (United States)

Ling, Jing; Wu, Xiaoli; Fu, Ziyi; Tan, Jie; Xu, Qing

2015-10-01

Our previous study showed that the expression of miR-197 in leiomyoma was down-regulated compared with myometrium. Further, miR-197 has been identified to affect uterine leiomyoma cell proliferation, apoptosis, and metastasis ability, though the responsible molecular mechanism has not been well elucidated. In this study, we sought to determine the expression patterns of miR-197 targeted genes and to explore their potential functions, participating Pathways and the networks that are involved in the biological behavior of human uterine leiomyoma. After transfection of human uterine leiomyoma cells with miR-197, we confirmed the expression level of miR-197 using quantitative real-time PCR (qRT-PCR), and we detected the gene expression profiles after miR-197 over-expression through DNA microarray analysis. Further, we performed GO and Pathway analysis. The dominantly dys-regulated genes, which were up- or down-regulated by more than 10-fold, compared with parental cells, were confirmed using qRT-PCR technology. Compared with the control group, miR-197 was up-regulated by 30-fold after miR-197 lentiviral transfection. The microarray data showed that 872 genes were dys-regulated by more than 2-fold in human uterine leiomyoma cells after miR-197 overexpression, including 537 up-regulated and 335 down-regulated genes. The GO analysis indicated that the dys-regulated genes were primarily involved in response to stimuli, multicellular organ processes, and the signaling of biological progression. Further, Pathway analysis data showed that these genes participated in regulating several signaling Pathways, including the JAK/STAT signaling Pathway, the Toll-like receptor signaling Pathway, and cytokine-cytokine receptor interaction. The qRT-PCR results confirmed that 17 of the 66 selected genes, which were up- or down-regulated more than 10-fold by miR-197, were consistent with the microarray results, including tumorigenesis-related genes, such as DRT7, SLC549, SFMBT2, FLJ37956

RT-qPCR normalization genes in the red alga Chondrus crispus.

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Nathalie Kowalczyk

Full Text Available Chondrus crispus is a common red macroalga living on the rocky shores of the North Atlantic Ocean. It has a long research history, being a major source of carrageenan, a thickener widely used in the food industry, but also for physiological and ecological studies. To establish it as a model for red algae, its genome has been sequenced, allowing the development of molecular tools such as quantification of gene expression, including RNAseq and RT-qPCR. To determine appropriate genes for RT-qPCR normalization, the expression of 14 genes was monitored in 18 conditions using two sets of algal samples: samples from the sequenced strain, cultured and stressed in laboratory conditions and C. crispus collected on the shore and stressed in situ. The expression stability of the genes between the samples was evaluated by comparing the Ct range and using the programs geNorm and NormFinder. The candidate genes encoded translation related proteins (initiation factors IF4A-1 and IF4A-2, elongation factor EF1α and eRF3, an eukaryotic polypeptide chain release factor, cytoskeleton proteins (two β-tubulins, α-tubulin and actin, enzymes involved in the pentose phosphate pathway (glucose 6-phosphate deshydrogenase, protein recycling process (ubiquitin and ubiquitin-conjugating enzyme and glycolysis (isocitrate dehydrogenase. The two sets of samples showed different expression patterns. Most of the genes were stable in the algae cultivated in the laboratory, whereas environmental samples showed a more important variation in gene expression. When analyzing the two sets separately, the ranking of the most stables genes were different from one method to another. When considering all samples, the two statistical methods were concordant, revealing translation initiation factor 4A-2 and eukaryotic polypeptide chain release factor 3 as pertinent normalization genes. This study highlights thus the importance of testing reference genes according to the experiments as well

Sequence polymorphism can produce serious artefacts in real-time PCR assays: hard lessons from Pacific oysters

Directory of Open Access Journals (Sweden)

Camara Mark D

2008-05-01

Full Text Available Abstract Background Since it was first described in the mid-1990s, quantitative real time PCR (Q-PCR has been widely used in many fields of biomedical research and molecular diagnostics. This method is routinely used to validate whole transcriptome analyses such as DNA microarrays, suppressive subtractive hybridization (SSH or differential display techniques such as cDNA-AFLP (Amplification Fragment Length Polymorphism. Despite efforts to optimize the methodology, misleading results are still possible, even when standard optimization approaches are followed. Results As part of a larger project aimed at elucidating transcriptome-level responses of Pacific oysters (Crassostrea gigas to various environmental stressors, we used microarrays and cDNA-AFLP to identify Expressed Sequence Tag (EST fragments that are differentially expressed in response to bacterial challenge in two heat shock tolerant and two heat shock sensitive full-sib oyster families. We then designed primers for these differentially expressed ESTs in order to validate the results using Q-PCR. For two of these ESTs we tested fourteen primer pairs each and using standard optimization methods (i.e. melt-curve analysis to ensure amplification of a single product, determined that of the fourteen primer pairs tested, six and nine pairs respectively amplified a single product and were thus acceptable for further testing. However, when we used these primers, we obtained different statistical outcomes among primer pairs, raising unexpected but serious questions about their reliability. We hypothesize that as a consequence of high levels of sequence polymorphism in Pacific oysters, Q-PCR amplification is sub-optimal in some individuals because sequence variants in priming sites results in poor primer binding and amplification in some individuals. This issue is similar to the high frequency of null alleles observed for microsatellite markers in Pacific oysters. Conclusion This study highlights

Selection of reference genes for quantitative gene expression normalization in flax (Linum usitatissimum L.

Directory of Open Access Journals (Sweden)

Neutelings Godfrey

2010-04-01

Full Text Available Abstract Background Quantitative real-time PCR (qRT-PCR is currently the most accurate method for detecting differential gene expression. Such an approach depends on the identification of uniformly expressed 'housekeeping genes' (HKGs. Extensive transcriptomic data mining and experimental validation in different model plants have shown that the reliability of these endogenous controls can be influenced by the plant species, growth conditions and organs/tissues examined. It is therefore important to identify the best reference genes to use in each biological system before using qRT-PCR to investigate differential gene expression. In this paper we evaluate different candidate HKGs for developmental transcriptomic studies in the economically-important flax fiber- and oil-crop (Linum usitatissimum L. Results Specific primers were designed in order to quantify the expression levels of 20 different potential housekeeping genes in flax roots, internal- and external-stem tissues, leaves and flowers at different developmental stages. After calculations of PCR efficiencies, 13 HKGs were retained and their expression stabilities evaluated by the computer algorithms geNorm and NormFinder. According to geNorm, 2 Transcriptional Elongation Factors (TEFs and 1 Ubiquitin gene are necessary for normalizing gene expression when all studied samples are considered. However, only 2 TEFs are required for normalizing expression in stem tissues. In contrast, NormFinder identified glyceraldehyde-3-phosphate dehydrogenase (GADPH as the most stably expressed gene when all samples were grouped together, as well as when samples were classed into different sub-groups. qRT-PCR was then used to investigate the relative expression levels of two splice variants of the flax LuMYB1 gene (homologue of AtMYB59. LuMYB1-1 and LuMYB1-2 were highly expressed in the internal stem tissues as compared to outer stem tissues and other samples. This result was confirmed with both ge