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.

Evaluation of Suitable Reference Genes for Normalization of qPCR Gene Expression Studies in Brinjal (Solanum melongena L.) During Fruit Developmental Stages.

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Kanakachari, Mogilicherla; Solanke, Amolkumar U; Prabhakaran, Narayanasamy; Ahmad, Israr; Dhandapani, Gurusamy; Jayabalan, Narayanasamy; Kumar, Polumetla Ananda

2016-02-01

Brinjal/eggplant/aubergine is one of the major solanaceous vegetable crops. Recent availability of genome information greatly facilitates the fundamental research on brinjal. Gene expression patterns during different stages of fruit development can provide clues towards the understanding of its biological functions. Quantitative real-time PCR (qPCR) has become one of the most widely used methods for rapid and accurate quantification of gene expression. However, its success depends on the use of a suitable reference gene for data normalization. For qPCR analysis, a single reference gene is not universally suitable for all experiments. Therefore, reference gene validation is a crucial step. Suitable reference genes for qPCR analysis of brinjal fruit development have not been investigated so far. In this study, we have selected 21 candidate reference genes from the Brinjal (Solanum melongena) Plant Gene Indices database (compbio.dfci.harvard.edu/tgi/plant.html) and studied their expression profiles by qPCR during six different fruit developmental stages (0, 5, 10, 20, 30, and 50 days post anthesis) along with leaf samples of the Pusa Purple Long (PPL) variety. To evaluate the stability of gene expression, geNorm and NormFinder analytical softwares were used. geNorm identified SAND (SAND family protein) and TBP (TATA binding protein) as the best pairs of reference genes in brinjal fruit development. The results showed that for brinjal fruit development, individual or a combination of reference genes should be selected for data normalization. NormFinder identified Expressed gene (expressed sequence) as the best single reference gene in brinjal fruit development. In this study, we have identified and validated for the first time reference genes to provide accurate transcript normalization and quantification at various fruit developmental stages of brinjal which can also be useful for gene expression studies in other Solanaceae plant species.

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.

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.

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.

Analysis of the stability of housekeeping gene expression in the left cardiac ventricle of rats submitted to chronic intermittent hypoxia

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Guilherme Silva Julian

Full Text Available ABSTRACT Obstructive sleep apnea (OSA has been associated with oxidative stress and various cardiovascular consequences, such as increased cardiovascular disease risk. Quantitative real-time PCR is frequently employed to assess changes in gene expression in experimental models. In this study, we analyzed the effects of chronic intermittent hypoxia (an experimental model of OSA on housekeeping gene expression in the left cardiac ventricle of rats. Analyses via four different approaches-use of the geNorm, BestKeeper, and NormFinder algorithms; and 2−ΔCt (threshold cycle data analysis-produced similar results: all genes were found to be suitable for use, glyceraldehyde-3-phosphate dehydrogenase and 18S being classified as the most and the least stable, respectively. The use of more than one housekeeping gene is strongly advised.

Transcriptional expression of type I interferon response genes and stability of housekeeping genes in the human endometrium and endometriosis

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Vestergaard, Anna L; Knudsen, Ulla B; Munk, Torben

2011-01-01

Endometriosis is a painful chronic female disease defined by the presence of endometrial tissue implants in ectopic locations. The pathogenesis is much debated, and type I interferons could be involved. The expression of genes of the type I interferon response were profiled by a specific PCR Array...... of RNA obtained from ectopic and eutopic endometrium collected from 9 endometriosis patients and 9 healthy control women. Transcriptional expression levels of selected interferon-regulated and housekeeping genes were investigated by real-time quantitative reverse transcriptase PCR (qRT-PCR). Stably...... expressed housekeeping genes for valid normalization of transcriptional studies of endometrium and endometriosis have not yet been published. Here, seven housekeeping genes were evaluated for stability using the GeNorm and NormFinder software. A normalization factor based on HMBS, TBP, and YWHAZ expression...

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

Validation of housekeeping genes in the brains of rats submitted to chronic intermittent hypoxia, a sleep apnea model.

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Julian, Guilherme Silva; de Oliveira, Renato Watanabe; Perry, Juliana Cini; Tufik, Sergio; Chagas, Jair Ribeiro

2014-01-01

Obstructive sleep apnea (OSA) is a syndrome characterized by intermittent nocturnal hypoxia, sleep fragmentation, hypercapnia and respiratory effort, and it has been associated with several complications, such as diabetes, hypertension and obesity. Quantitative real-time PCR has been performed in previous OSA-related studies; however, these studies were not validated using proper reference genes. We have examined the effects of chronic intermittent hypoxia (CIH), which is an experimental model mainly of cardiovascular consequences of OSA, on reference genes, including beta-actin, beta-2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase, hypoxanthine guanine phosphoribosyl transferase and eukaryotic 18S rRNA, in different areas of the brain. All stability analyses were performed using the geNorm, Normfinder and BestKeeper software programs. With exception of the 18S rRNA, all of the evaluated genes were shown to be stable following CIH exposure. However, gene stability rankings were dependent on the area of the brain that was analyzed and varied according to the software that was used. This study demonstrated that CIH affects various brain structures differently. With the exception of the 18S rRNA, all of the tested genes are suitable for use as housekeeping genes in expression analyses.

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

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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.

The Construction and Norm-Finding of a Rating Scale for Diagnosing Attention Deficit Hyperactivity Disorder in Children

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Zahra Hooshyari

2008-01-01

Full Text Available Objective: Evaluation and diagnosis are determinants of treatment outcome; therefore, diagnostic tools should be accurate. The purpose of the present research is construction and norm-finding of a rating scale for the diagnosis of attention deficit hyperactivity disorder (ADHD. Method: A checklist based on behavioral symptoms of ADHD from clinical sources was constructed, based on interview with patients and the medical records. After being studied by specialists, this checklist was reduced to items and arranged in a rating scale format. The study population included Tehran's elementary school students (boys and girls from which a sample of 800 children was chosen in a random-cluster manner. Then teachers were asked to rate the students according to the scale characteristics. After two weeks, the subjects were tested and interviewed using a corrected form of the scale, the rating scale of Swanson, Nolan and Pelham (SNAP-IV and Diagnostic and Statistical Manual of Mental Disorders (DSM-IV. After data analysis the scale was reduced to 23 items. Results: According to the results of the analysis, the scale had two factors: The first factor was hyperactivity that explained 37.41%, and the second factor was attention deficit that explained 33.78% of the total variance . Cronbach's alpha was 0.96 and the alpha of the two subscales, hyperactivity and attention deficit, was 0.94 and 0.96, respectively. Spearman-Brown Coefficient was 0.78 and scale coefficient correlation with a similar from (SNAP-IV was 0.82. Criterion validity coefficient of the scale was 0.76 and the content validity of the scale was confirmed by the experts. According to sensitivity analysis of the trait, the cutoff point of the scale was 35. Considering the above findings, we can safely use the above scale in clinical studies. Conclusion: Considering the obtained values in the psychometric indexes and the consensus among specialists on the basis of test content validity and

Use of Maximum Likelihood-Mixed Models to select stable reference genes: a case of heat stress response in sheep

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Salces Judit

2011-08-01

Full Text Available Abstract Background Reference genes with stable expression are required to normalize expression differences of target genes in qPCR experiments. Several procedures and companion software have been proposed to find the most stable genes. Model based procedures are attractive because they provide a solid statistical framework. NormFinder, a widely used software, uses a model based method. The pairwise comparison procedure implemented in GeNorm is a simpler procedure but one of the most extensively used. In the present work a statistical approach based in Maximum Likelihood estimation under mixed models was tested and compared with NormFinder and geNorm softwares. Sixteen candidate genes were tested in whole blood samples from control and heat stressed sheep. Results A model including gene and treatment as fixed effects, sample (animal, gene by treatment, gene by sample and treatment by sample interactions as random effects with heteroskedastic residual variance in gene by treatment levels was selected using goodness of fit and predictive ability criteria among a variety of models. Mean Square Error obtained under the selected model was used as indicator of gene expression stability. Genes top and bottom ranked by the three approaches were similar; however, notable differences for the best pair of genes selected for each method and the remaining genes of the rankings were shown. Differences among the expression values of normalized targets for each statistical approach were also found. Conclusions Optimal statistical properties of Maximum Likelihood estimation joined to mixed model flexibility allow for more accurate estimation of expression stability of genes under many different situations. Accurate selection of reference genes has a direct impact over the normalized expression values of a given target gene. This may be critical when the aim of the study is to compare expression rate differences among samples under different environmental

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.

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.

Screening and Validation of Housekeeping Genes of the Root and Cotyledon of Cunninghamia lanceolata under Abiotic Stresses by Using Quantitative Real-Time PCR

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Wenlong Bao

2016-07-01

Full Text Available Cunninghamia lanceolata (Chinese fir is a fast-growing and commercially important conifer of the Cupressaceae family. Due to the unavailability of complete genome sequences and relatively poor genetic background information of the Chinese fir, it is necessary to identify and analyze the expression levels of suitable housekeeping genes (HKGs as internal reference for precise analysis. Based on the results of database analysis and transcriptome sequencing, we have chosen five candidate HKGs (Actin, GAPDH, EF1a, 18S rRNA, and UBQ with conservative sequences in the Chinese fir and related species for quantitative analysis. The expression levels of these HKGs in roots and cotyledons under five different abiotic stresses in different time intervals were measured by qRT-PCR. The data were statistically analyzed using the following algorithms: NormFinder, BestKeeper, and geNorm. Finally, RankAggreg was applied to merge the sequences generated from three programs and rank these according to consensus sequences. The expression levels of these HKGs showed variable stabilities under different abiotic stresses. Among these, Actin was the most stable internal control in root, and GAPDH was the most stable housekeeping gene in cotyledon. We have also described an experimental procedure for selecting HKGs based on the de novo sequencing database of other non-model plants.

rpb2 is a reliable reference gene for quantitative gene expression analysis in the dermatophyte Trichophyton rubrum.

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Jacob, Tiago R; Peres, Nalu T A; Persinoti, Gabriela F; Silva, Larissa G; Mazucato, Mendelson; Rossi, Antonio; Martinez-Rossi, Nilce M

2012-05-01

The selection of reference genes used for data normalization to quantify gene expression by real-time PCR amplifications (qRT-PCR) is crucial for the accuracy of this technique. In spite of this, little information regarding such genes for qRT-PCR is available for gene expression analyses in pathogenic fungi. Thus, we investigated the suitability of eight candidate reference genes in isolates of the human dermatophyte Trichophyton rubrum subjected to several environmental challenges, such as drug exposure, interaction with human nail and skin, and heat stress. The stability of these genes was determined by geNorm, NormFinder and Best-Keeper programs. The gene with the most stable expression in the majority of the conditions tested was rpb2 (DNA-dependent RNA polymerase II), which was validated in three T. rubrum strains. Moreover, the combination of rpb2 and chs1 (chitin synthase) genes provided for the most reliable qRT-PCR data normalization in T. rubrum under a broad range of biological conditions. To the best of our knowledge this is the first report on the selection of reference genes for qRT-PCR data normalization in dermatophytes and the results of these studies should permit further analysis of gene expression under several experimental conditions, with improved accuracy and reliability.

Assessing reference genes for accurate transcript normalization using quantitative real-time PCR in pearl millet [Pennisetum glaucum (L. R. Br].

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Prasenjit Saha

Full Text Available Pearl millet [Pennisetum glaucum (L. R.Br.], a close relative of Panicoideae food crops and bioenergy grasses, offers an ideal system to perform functional genomics studies related to C4 photosynthesis and abiotic stress tolerance. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR provides a sensitive platform to conduct such gene expression analyses. However, the lack of suitable internal control reference genes for accurate transcript normalization during qRT-PCR analysis in pearl millet is the major limitation. Here, we conducted a comprehensive assessment of 18 reference genes on 234 samples which included an array of different developmental tissues, hormone treatments and abiotic stress conditions from three genotypes to determine appropriate reference genes for accurate normalization of qRT-PCR data. Analyses of Ct values using Stability Index, BestKeeper, ΔCt, Normfinder, geNorm and RefFinder programs ranked PP2A, TIP41, UBC2, UBQ5 and ACT as the most reliable reference genes for accurate transcript normalization under different experimental conditions. Furthermore, we validated the specificity of these genes for precise quantification of relative gene expression and provided evidence that a combination of the best reference genes are required to obtain optimal expression patterns for both endogeneous genes as well as transgenes in pearl millet.

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.

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.

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

Selection of reference genes for quantitative gene expression normalization in flax (Linum usitatissimum L.

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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

Selection of reference genes for quantitative gene expression normalization in flax (Linum usitatissimum L.).

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Huis, Rudy; Hawkins, Simon; Neutelings, Godfrey

2010-04-19

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). 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 geNorm-designated- and Norm

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

Reference miRNAs for miRNAome analysis of urothelial carcinomas.

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Nadine Ratert

Full Text Available BACKGROUND/OBJECTIVE: Reverse transcription quantitative real-time PCR (RT-qPCR is widely used in microRNA (miRNA expression studies on cancer. To compensate for the analytical variability produced by the multiple steps of the method, relative quantification of the measured miRNAs is required, which is based on normalization to endogenous reference genes. No study has been performed so far on reference miRNAs for normalization of miRNA expression in urothelial carcinoma. The aim of this study was to identify suitable reference miRNAs for miRNA expression studies by RT-qPCR in urothelial carcinoma. METHODS: Candidate reference miRNAs were selected from 24 urothelial carcinoma and normal bladder tissue samples by miRNA microarrays. The usefulness of these candidate reference miRNAs together with the commonly for normalization purposes used small nuclear RNAs RNU6B, RNU48, and Z30 were thereafter validated by RT-qPCR in 58 tissue samples and analyzed by the algorithms geNorm, NormFinder, and BestKeeper. PRINCIPAL FINDINGS: Based on the miRNA microarray data, a total of 16 miRNAs were identified as putative reference genes. After validation by RT-qPCR, miR-101, miR-125a-5p, miR-148b, miR-151-5p, miR-181a, miR-181b, miR-29c, miR-324-3p, miR-424, miR-874, RNU6B, RNU48, and Z30 were used for geNorm, NormFinder, and BestKeeper analyses that gave different combinations of recommended reference genes for normalization. CONCLUSIONS: The present study provided the first systematic analysis for identifying suitable reference miRNAs for miRNA expression studies of urothelial carcinoma by RT-qPCR. Different combinations of reference genes resulted in reliable expression data for both strongly and less strongly altered miRNAs. Notably, RNU6B, which is the most frequently used reference gene for miRNA studies, gave inaccurate normalization. The combination of four (miR-101, miR-125a-5p, miR-148b, and miR-151-5p or three (miR-148b, miR-181b, and miR-874

Identification of valid reference genes for gene expression studies of human stomach cancer by reverse transcription-qPCR

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Rho, Hyun-Wook; Lee, Byoung-Chan; Choi, Eun-Seok; Choi, Il-Ju; Lee, Yeon-Su; Goh, Sung-Ho

2010-01-01

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a powerful method for the analysis of gene expression. Target gene expression levels are usually normalized to a consistently expressed reference gene also known as internal standard, in the same sample. However, much effort has not been expended thus far in the search for reference genes suitable for the study of stomach cancer using RT-qPCR, although selection of optimal reference genes is critical for interpretation of results. We assessed the suitability of six possible reference genes, beta-actin (ACTB), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyl transferase 1 (HPRT1), beta-2-microglobulin (B2M), ribosomal subunit L29 (RPL29) and 18S ribosomal RNA (18S rRNA) in 20 normal and tumor stomach tissue pairs of stomach cancer patients and 6 stomach cancer cell lines, by RT-qPCR. Employing expression stability analyses using NormFinder and geNorm algorithms we determined the order of performance of these reference genes and their variation values. This RT-qPCR study showed that there are statistically significant (p < 0.05) differences in the expression levels of HPRT1 and 18S rRNA in 'normal-' versus 'tumor stomach tissues'. The stability analyses by geNorm suggest B2M-GAPDH, as best reference gene combination for 'stomach cancer cell lines'; RPL29-HPRT1, for 'all stomach tissues'; and ACTB-18S rRNA, for 'all stomach cell lines and tissues'. NormFinder also identified B2M as the best reference gene for 'stomach cancer cell lines', RPL29-B2M for 'all stomach tissues', and 18S rRNA-ACTB for 'all stomach cell lines and tissues'. The comparisons of normalized expression of the target gene, GPNMB, showed different interpretation of target gene expression depend on best single reference gene or combination. This study validated RPL29 and RPL29-B2M as the best single reference

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

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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

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.

Validation and comparison of reference genes for qPCR normalization of celery (Apium graveolens at different development stages

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Meng-Yao eLi

2016-03-01

Full Text Available A suitable reference gene is an important prerequisite for guarantying accurate and reliable results in qPCR analysis. Celery is one of the representative vegetable in Apiaceae and is widely cultivated and consumed in the world. However, no reports have been previously published concerning reference genes in celery. In this study, the expression stabilities of nine candidate reference genes in leaf blade and petiole at different development stages were evaluated using three statistics algorithms geNorm, NormFinder, and BestKeeper. Our results showed that TUB-B, TUB-A, and UBC were the most reference genes among all tested samples. GAPDH represented the maximum stability for most individual sample, while the UBQ displayed the minimum stability. To further validate the stability of reference genes, the expression pattern of AgAP2-2 was calculated by using the selected genes for normalization. In addition, the expression patterns of several development-related genes were studied using the selected reference gene. Our results will be beneficial for further studies on gene transcription in celery.

Reference Gene Selection for qRT-PCR Normalization Analysis in kenaf (Hibiscus cannabinus L. under Abiotic Stress and Hormonal Stimuli

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Xiaoping Niu

2017-05-01

Full Text Available Kenaf (Hibiscus cannabinus L., an environmental friendly and economic fiber crop, has a certain tolerance to abiotic stresses. Identification of reliable reference genes for transcript normalization of stress responsive genes expression by quantitative real-time PCR (qRT-PCR is important for exploring the molecular mechanisms of plants response to abiotic stresses. In this study, nine candidate reference genes were cloned, and their expression stabilities were assessed in 132 abiotic stress and hormonal stimuli samples of kenaf using geNorm, NormFinder, and BestKeeper algorithms. Results revealed that HcPP2A (Protein phosphatase 2A and HcACT7 (Actin 7 were the optimum reference genes across all samples; HcUBC (Ubiquitin-conjugating enzyme like protein was the worst reference gene for transcript normalization. The reliability of the selected reference genes was further confirmed by evaluating the expression profile of HcWRKY28 gene at different stress durations. This work will benefit future studies on discovery of stress-tolerance genes and stress-signaling pathways in this important fiber crop.

Whole-Transcriptome Selection and Evaluation of Internal Reference Genes for Expression Analysis in Protocorm Development of Dendrobium officinale Kimura et Migo.

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Hongqiang An

Full Text Available Dendrobium officinale Kimu et Migo has increased many researchers' interest for its high medical and horticultural values and the molecular mechanism of its protocorm development remains unclear. In this study, 19 genes from 26 most stably expressed genes in whole transcriptome of protocorms and 5 housekeeping genes were used as candidate reference genes and screened with 4 application softwares (geNorm, NormFinder, BestKeeper and RefFinder. The results showed that a few reference genes could effectively normalize expression level of specific genes in protocorm development and the optimal top 2 reference genes were ASS and APH1L. Meanwhile, validation of GNOM, AP2 and temperature induced gene (TIL for normalization demonstrates the usefulness of the validated candidate reference genes. The expression profiles of these genes varied under protocorms and temperature stress according to the stablest and unstablest reference genes, which proved the importance of the choice of appropriate reference genes. The first systematic evaluation of stably expressed genes will be very useful in the future analysis of specific genes expression in D. officinale.

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.

Identification of Appropriate Reference Genes for Normalization of miRNA Expression in Grafted Watermelon Plants under Different Nutrient Stresses.

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Wu, Weifang; Deng, Qin; Shi, Pibiao; Yang, Jinghua; Hu, Zhongyuan; Zhang, Mingfang

2016-01-01

Watermelon (Citrullus lanatus) is a globally important crop belonging to the family Cucurbitaceae. The grafting technique is commonly used to improve its tolerance to stress, as well as to enhance its nutrient uptake and utilization. It is believed that miRNA is most likely involved in its nutrient-starvation response as a graft-transportable signal. The quantitative real-time reverse transcriptase polymerase chain reaction is the preferred method for miRNA functional analysis, in which reliable reference genes for normalization are crucial to ensure the accuracy. The purpose of this study was to select appropriate reference genes in scion (watermelon) and rootstocks (squash and bottle gourd) of grafted watermelon plants under normal growth conditions and nutrient stresses (nitrogen and phosphorus starvation). Under nutrient starvation, geNorm identified miR167c and miR167f as two most stable genes in both watermelon leaves and squash roots. miR166b was recommended by both geNorm and NormFinder as the best reference in bottle gourd roots under nutrient limitation. Expression of a new Cucurbitaceae miRNA, miR85, was used to validate the reliability of candidate reference genes under nutrient starvation. Moreover, by comparing several target genes expression in qRT-PCR analysis with those in RNA-seq data, miR166b and miR167c were proved to be the most suitable reference genes to normalize miRNA expression under normal growth condition in scion and rootstock tissues, respectively. This study represents the first comprehensive survey of the stability of miRNA reference genes in Cucurbitaceae and provides valuable information for investigating more accurate miRNA expression involving grafted watermelon plants.

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 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.

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.

Reference genes for accurate transcript normalization in citrus genotypes under different experimental conditions.

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Valéria Mafra

Full Text Available Real-time reverse transcription PCR (RT-qPCR has emerged as an accurate and widely used technique for expression profiling of selected genes. However, obtaining reliable measurements depends on the selection of appropriate reference genes for gene expression normalization. The aim of this work was to assess the expression stability of 15 candidate genes to determine which set of reference genes is best suited for transcript normalization in citrus in different tissues and organs and leaves challenged with five pathogens (Alternaria alternata, Phytophthora parasitica, Xylella fastidiosa and Candidatus Liberibacter asiaticus. We tested traditional genes used for transcript normalization in citrus and orthologs of Arabidopsis thaliana genes described as superior reference genes based on transcriptome data. geNorm and NormFinder algorithms were used to find the best reference genes to normalize all samples and conditions tested. Additionally, each biotic stress was individually analyzed by geNorm. In general, FBOX (encoding a member of the F-box family and GAPC2 (GAPDH was the most stable candidate gene set assessed under the different conditions and subsets tested, while CYP (cyclophilin, TUB (tubulin and CtP (cathepsin were the least stably expressed genes found. Validation of the best suitable reference genes for normalizing the expression level of the WRKY70 transcription factor in leaves infected with Candidatus Liberibacter asiaticus showed that arbitrary use of reference genes without previous testing could lead to misinterpretation of data. Our results revealed FBOX, SAND (a SAND family protein, GAPC2 and UPL7 (ubiquitin protein ligase 7 to be superior reference genes, and we recommend their use in studies of gene expression in citrus species and relatives. This work constitutes the first systematic analysis for the selection of superior reference genes for transcript normalization in different citrus organs and under biotic stress.

Selection of reference genes for expression analysis in the entomophthoralean fungus Pandora neoaphidis.

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Chen, Chun; Xie, Tingna; Ye, Sudan; Jensen, Annette Bruun; Eilenberg, Jørgen

2016-01-01

The selection of suitable reference genes is crucial for accurate quantification of gene expression and can add to our understanding of host-pathogen interactions. To identify suitable reference genes in Pandora neoaphidis, an obligate aphid pathogenic fungus, the expression of three traditional candidate genes including 18S rRNA(18S), 28S rRNA(28S) and elongation factor 1 alpha-like protein (EF1), were measured by quantitative polymerase chain reaction at different developmental stages (conidia, conidia with germ tubes, short hyphae and elongated hyphae), and under different nutritional conditions. We calculated the expression stability of candidate reference genes using four algorithms including geNorm, NormFinder, BestKeeper and Delta Ct. The analysis results revealed that the comprehensive ranking of candidate reference genes from the most stable to the least stable was 18S (1.189), 28S (1.414) and EF1 (3). The 18S was, therefore, the most suitable reference gene for real-time RT-PCR analysis of gene expression under all conditions. These results will support further studies on gene expression in P. neoaphidis. Copyright © 2015 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Selection of reference genes for expression analysis in the entomophthoralean fungus Pandora neoaphidis

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Chun Chen

2016-03-01

Full Text Available Abstract The selection of suitable reference genes is crucial for accurate quantification of gene expression and can add to our understanding of host–pathogen interactions. To identify suitable reference genes in Pandora neoaphidis, an obligate aphid pathogenic fungus, the expression of three traditional candidate genes including 18S rRNA(18S, 28S rRNA(28S and elongation factor 1 alpha-like protein (EF1, were measured by quantitative polymerase chain reaction at different developmental stages (conidia, conidia with germ tubes, short hyphae and elongated hyphae, and under different nutritional conditions. We calculated the expression stability of candidate reference genes using four algorithms including geNorm, NormFinder, BestKeeper and Delta Ct. The analysis results revealed that the comprehensive ranking of candidate reference genes from the most stable to the least stable was 18S (1.189, 28S (1.414 and EF1 (3. The 18S was, therefore, the most suitable reference gene for real-time RT-PCR analysis of gene expression under all conditions. These results will support further studies on gene expression in P. neoaphidis.

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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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.

Selection of suitable reference genes for RT-qPCR analyses in cyanobacteria.

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Filipe Pinto

Full Text Available Cyanobacteria are a group of photosynthetic prokaryotes that have a diverse morphology, minimal nutritional requirements and metabolic plasticity that has made them attractive organisms to use in biotechnological applications. The use of these organisms as cell factories requires the knowledge of their physiology and metabolism at a systems level. For the quantification of gene transcripts real-time quantitative polymerase chain reaction (RT-qPCR is the standard technique. However, to obtain reliable RT-qPCR results the use and validation of reference genes is mandatory. Towards this goal we have selected and analyzed twelve candidate reference genes from three morphologically distinct cyanobacteria grown under routinely used laboratory conditions. The six genes exhibiting less variation in each organism were evaluated in terms of their expression stability using geNorm, NormFinder and BestKeeper. In addition, the minimum number of reference genes required for normalization was determined. Based on the three algorithms, we provide a list of genes for cyanobacterial RT-qPCR data normalization. To our knowledge, this is the first work on the validation of reference genes for cyanobacteria constituting a valuable starting point for future works.

Selection of reference genes is critical for miRNA expression analysis in human cardiac tissue. A focus on atrial fibrillation.

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Masè, Michela; Grasso, Margherita; Avogaro, Laura; D'Amato, Elvira; Tessarolo, Francesco; Graffigna, Angelo; Denti, Michela Alessandra; Ravelli, Flavia

2017-01-24

MicroRNAs (miRNAs) are emerging as key regulators of complex biological processes in several cardiovascular diseases, including atrial fibrillation (AF). Reverse transcription-quantitative polymerase chain reaction is a powerful technique to quantitatively assess miRNA expression profile, but reliable results depend on proper data normalization by suitable reference genes. Despite the increasing number of studies assessing miRNAs in cardiac disease, no consensus on the best reference genes has been reached. This work aims to assess reference genes stability in human cardiac tissue with a focus on AF investigation. We evaluated the stability of five reference genes (U6, SNORD48, SNORD44, miR-16, and 5S) in atrial tissue samples from eighteen cardiac-surgery patients in sinus rhythm and AF. Stability was quantified by combining BestKeeper, delta-C q , GeNorm, and NormFinder statistical tools. All methods assessed SNORD48 as the best and U6 as the worst reference gene. Applications of different normalization strategies significantly impacted miRNA expression profiles in the study population. Our results point out the necessity of a consensus on data normalization in AF studies to avoid the emergence of divergent biological conclusions.

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.

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

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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.

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.

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.

Selection of reliable reference genes in Caenorhabditis elegans for analysis of nanotoxicity.

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Zhang, Yanqiong; Chen, Dongliang; Smith, Michael A; Zhang, Baohong; Pan, Xiaoping

2012-01-01

Despite rapid development and application of a wide range of manufactured metal oxide nanoparticles (NPs), the understanding of potential risks of using NPs is less completed, especially at the molecular level. The nematode Caenorhabditis elegans (C.elegans) has been emerging as an environmental model to study the molecular mechanism of environmental contaminations, using standard genetic tools such as the real-time quantitative PCR (RT-qPCR). The most important factor that may affect the accuracy of RT-qPCR is to choose appropriate genes for normalization. In this study, we selected 13 reference gene candidates (act-1, cdc-42, pmp-3, eif-3.C, actin, act-2, csq-1, Y45F10D.4, tba-1, mdh-1, ama-1, F35G12.2, and rbd-1) to test their expression stability under different doses of nano-copper oxide (CuO 0, 1, 10, and 50 µg/mL) using RT-qPCR. Four algorithms, geNorm, NormFinder, BestKeeper, and the comparative ΔCt method, were employed to evaluate these 13 candidates expressions. As a result, tba-1, Y45F10D.4 and pmp-3 were the most reliable, which may be used as reference genes in future study of nanoparticle-induced genetic response using C.elegans.

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 Appropriate Reference Genes for Reverse Transcription-Quantitative PCR Studies in Different Tissues of a Desert Poplar via Comparision of Different Algorithms

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Hou-Ling Wang

2015-08-01

Full Text Available Despite the unshakable status of reverse transcription-quantitative PCR in gene expression analysis, it has certain disadvantages, including that the results are highly dependent on the reference genes selected for data normalization. Since inappropriate endogenous control genes will lead to inaccurate target gene expression profiles, the validation of suitable internal reference genes is essential. Given the increasing interest in functional genes and genomics of Populus euphratica, a desert poplar showing extraordinary adaptation to salt stress, we evaluated the expression stability of ten candidate reference genes in P. euphratica roots, stems, and leaves under salt stress conditions. We used five algorithms, namely, ΔCt, NormFinder, geNorm, GrayNorm, and a rank aggregation method (RankAggreg to identify suitable normalizers. To support the suitability of the identified reference genes and to compare the relative merits of these different algorithms, we analyzed and compared the relative expression levels of nine P. euphratica functional genes in different tissues. Our results indicate that a combination of multiple reference genes recommended by GrayNorm algorithm (e.g., a combination of Actin, EF1α, GAPDH, RP, UBQ in root should be used instead of a single reference gene. These results are valuable for research of gene identification in different P. euphratica tissues.

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.

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.

Selection of reference genes for tissue/organ samples on day 3 fifth-instar larvae in silkworm, Bombyx mori.

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Wang, Genhong; Chen, Yanfei; Zhang, Xiaoying; Bai, Bingchuan; Yan, Hao; Qin, Daoyuan; Xia, Qingyou

2018-06-01

The silkworm, Bombyx mori, is one of the world's most economically important insect. Surveying variations in gene expression among multiple tissue/organ samples will provide clues for gene function assignments and will be helpful for identifying genes related to economic traits or specific cellular processes. To ensure their accuracy, commonly used gene expression quantification methods require a set of stable reference genes for data normalization. In this study, 24 candidate reference genes were assessed in 10 tissue/organ samples of day 3 fifth-instar B. mori larvae using geNorm and NormFinder. The results revealed that, using the combination of the expression of BGIBMGA003186 and BGIBMGA008209 was the optimum choice for normalizing the expression data of the B. mori tissue/organ samples. The most stable gene, BGIBMGA003186, is recommended if just one reference gene is used. Moreover, the commonly used reference gene encoding cytoplasmic actin was the least appropriate reference gene of the samples investigated. The reliability of the selected reference genes was further confirmed by evaluating the expression profiles of two cathepsin genes. Our results may be useful for future studies involving the quantification of relative gene expression levels of different tissue/organ samples in B. mori. © 2018 Wiley Periodicals, Inc.

Reference gene validation for qPCR in rat carotid body during postnatal development

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Carroll John L

2011-10-01

Full Text Available Abstract Background The carotid bodies are the main arterial oxygen chemoreceptors in mammals. Afferent neural output from the carotid bodies to brainstem respiratory and cardiovascular nuclei provides tonic input and mediates important protective responses to acute and chronic hypoxia. It is widely accepted that the selection of reference genes for mRNA normalization in quantitative real-time PCR must be validated for a given tissue and set of conditions. This is particularly important for studies in carotid body during early postnatal maturation as the arterial oxygen tension undergoes major changes from fetal to postnatal life, which may affect reference gene expression. In order to determine the most stable and suitable reference genes for the study of rat carotid body during development, six commonly used reference genes, β-actin, RPII (RNA polymerase II, PPIA (peptidyl-proyl-isomerase A, TBP (TATA-box binding protein, GAPDH, and 18s rRNA, were evaluated in two age groups (P0-1 and P14-16 under three environmental oxygen conditions (normoxia, chronic hypoxia and chronic hyperoxia using the three most commonly used software programs, geNorm, NormFinder and BestKeeper. Findings The three programs produced similar results but the reference gene rankings were not identical between programs or experimental conditions. Overall, 18s rRNA was the least stable reference gene for carotid body and, when hyperoxia and/or hypoxia conditions were included, actin was similarly unstable. Conclusions Reference or housekeeping gene expression for qPCR studies of carotid body during postnatal development may vary with developmental stage and environmental conditions. Selection of the best reference gene or combination of reference genes for carotid body development studies should take environmental conditions into account. Two commonly used reference genes, 18s rRNA and actin, may be unsuitable for studies of carotid body maturation, especially if the study

Reference gene selection for quantitative gene expression studies during biological invasions: A test on multiple genes and tissues in a model ascidian Ciona savignyi.

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Huang, Xuena; Gao, Yangchun; Jiang, Bei; Zhou, Zunchun; Zhan, Aibin

2016-01-15

As invasive species have successfully colonized a wide range of dramatically different local environments, they offer a good opportunity to study interactions between species and rapidly changing environments. Gene expression represents one of the primary and crucial mechanisms for rapid adaptation to local environments. Here, we aim to select reference genes for quantitative gene expression analysis based on quantitative Real-Time PCR (qRT-PCR) for a model invasive ascidian, Ciona savignyi. We analyzed the stability of ten candidate reference genes in three tissues (siphon, pharynx and intestine) under two key environmental stresses (temperature and salinity) in the marine realm based on three programs (geNorm, NormFinder and delta Ct method). Our results demonstrated only minor difference for stability rankings among the three methods. The use of different single reference gene might influence the data interpretation, while multiple reference genes could minimize possible errors. Therefore, reference gene combinations were recommended for different tissues - the optimal reference gene combination for siphon was RPS15 and RPL17 under temperature stress, and RPL17, UBQ and TubA under salinity treatment; for pharynx, TubB, TubA and RPL17 were the most stable genes under temperature stress, while TubB, TubA and UBQ were the best under salinity stress; for intestine, UBQ, RPS15 and RPL17 were the most reliable reference genes under both treatments. Our results suggest that the necessity of selection and test of reference genes for different tissues under varying environmental stresses. The results obtained here are expected to reveal mechanisms of gene expression-mediated invasion success using C. savignyi as a model species. Copyright © 2015 Elsevier B.V. All rights reserved.

Identification of Reference Genes for Quantitative Gene Expression Studies in a Non-Model Tree Pistachio (Pistacia vera L..

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Maryam Moazzam Jazi

Full Text Available The tree species, Pistacia vera (P. vera is an important commercial product that is salt-tolerant and long-lived, with a possible lifespan of over one thousand years. Gene expression analysis is an efficient method to explore the possible regulatory mechanisms underlying these characteristics. Therefore, having the most suitable set of reference genes is required for transcript level normalization under different conditions in P. vera. In the present study, we selected eight widely used reference genes, ACT, EF1α, α-TUB, β-TUB, GAPDH, CYP2, UBQ10, and 18S rRNA. Using qRT-PCR their expression was assessed in 54 different samples of three cultivars of P. vera. The samples were collected from different organs under various abiotic treatments (cold, drought, and salt across three time points. Several statistical programs (geNorm, NormFinder, and BestKeeper were applied to estimate the expression stability of candidate reference genes. Results obtained from the statistical analysis were then exposed to Rank aggregation package to generate a consensus gene rank. Based on our results, EF1α was found to be the superior reference gene in all samples under all abiotic treatments. In addition to EF1α, ACT and β-TUB were the second best reference genes for gene expression analysis in leaf and root. We recommended β-TUB as the second most stable gene for samples under the cold and drought treatments, while ACT holds the same position in samples analyzed under salt treatment. This report will benefit future research on the expression profiling of P. vera and other members of the Anacardiaceae family.

Identification of Reference Genes for Quantitative Gene Expression Studies in a Non-Model Tree Pistachio (Pistacia vera L.).

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Moazzam Jazi, Maryam; Ghadirzadeh Khorzoghi, Effat; Botanga, Christopher; Seyedi, Seyed Mahdi

2016-01-01

The tree species, Pistacia vera (P. vera) is an important commercial product that is salt-tolerant and long-lived, with a possible lifespan of over one thousand years. Gene expression analysis is an efficient method to explore the possible regulatory mechanisms underlying these characteristics. Therefore, having the most suitable set of reference genes is required for transcript level normalization under different conditions in P. vera. In the present study, we selected eight widely used reference genes, ACT, EF1α, α-TUB, β-TUB, GAPDH, CYP2, UBQ10, and 18S rRNA. Using qRT-PCR their expression was assessed in 54 different samples of three cultivars of P. vera. The samples were collected from different organs under various abiotic treatments (cold, drought, and salt) across three time points. Several statistical programs (geNorm, NormFinder, and BestKeeper) were applied to estimate the expression stability of candidate reference genes. Results obtained from the statistical analysis were then exposed to Rank aggregation package to generate a consensus gene rank. Based on our results, EF1α was found to be the superior reference gene in all samples under all abiotic treatments. In addition to EF1α, ACT and β-TUB were the second best reference genes for gene expression analysis in leaf and root. We recommended β-TUB as the second most stable gene for samples under the cold and drought treatments, while ACT holds the same position in samples analyzed under salt treatment. This report will benefit future research on the expression profiling of P. vera and other members of the Anacardiaceae family.

Identification and validation of reference genes for quantification of target gene expression with quantitative real-time PCR for tall fescue under four abiotic stresses.

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Zhimin Yang

Full Text Available Tall fescue (Festuca arundinacea Schreb. is widely utilized as a major forage and turfgrass species in the temperate regions of the world and is a valuable plant material for studying molecular mechanisms of grass stress tolerance due to its superior drought and heat tolerance among cool-season species. Selection of suitable reference genes for quantification of target gene expression is important for the discovery of molecular mechanisms underlying improved growth traits and stress tolerance. The stability of nine potential reference genes (ACT, TUB, EF1a, GAPDH, SAND, CACS, F-box, PEPKR1 and TIP41 was evaluated using four programs, GeNorm, NormFinder, BestKeeper, and RefFinder. The combinations of SAND and TUB or TIP41 and TUB were most stably expressed in salt-treated roots or leaves. The combinations of GAPDH with TIP41 or TUB were stable in roots and leaves under drought stress. TIP41 and PEPKR1 exhibited stable expression in cold-treated roots, and the combination of F-box, TIP41 and TUB was also stable in cold-treated leaves. CACS and TUB were the two most stable reference genes in heat-stressed roots. TIP41 combined with TUB and ACT was stably expressed in heat-stressed leaves. Finally, quantitative real-time polymerase chain reaction (qRT-PCR assays of the target gene FaWRKY1 using the identified most stable reference genes confirmed the reliability of selected reference genes. The selection of suitable reference genes in tall fescue will allow for more accurate identification of stress-tolerance genes and molecular mechanisms conferring stress tolerance in this stress-tolerant species.

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.

Selection of reference genes for quantitative real-time PCR expression studies of microdissected reproductive tissues in apomictic and sexual Boechera

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Amiteye Samuel

2011-08-01

Full Text Available Abstract Background Apomixis, a natural form of asexual seed production in plants, is considered to have great biotechnological potential for agriculture. It has been hypothesised that de-regulation of the sexual developmental pathway could trigger apomictic reproduction. The genus Boechera represents an interesting model system for understanding apomixis, having both sexual and apomictic genotypes at the diploid level. Quantitative qRT-PCR is the most extensively used method for validating genome-wide gene expression analyses, but in order to obtain reliable results, suitable reference genes are necessary. In this work we have evaluated six potential reference genes isolated from a 454 (FLX derived cDNA library of Boechera. RNA from live microdissected ovules and anthers at different developmental stages, as well as vegetative tissues of apomictic and sexual Boechera, were used to validate the candidates. Results Based on homologies with Arabidopsis, six genes were selected from a 454 cDNA library of Boechera: RPS18 (Ribosomal sub protein 18, Efalpha1 (Elongation factor 1 alpha, ACT 2 (Actin2, UBQ (polyubiquitin, PEX4 (Peroxisomal ubiquitin conjugating enzyme and At1g09770.1 (Arabidopsis thaliana cell division cycle 5. Total RNA was extracted from 17 different tissues, qRT-PCRs were performed, and raw Ct values were analyzed for primer efficiencies and gene ratios. The geNorm and normFinder applications were used for selecting the most stable genes among all tissues and specific tissue groups (ovule, anthers and vegetative tissues in both apomictic and sexual plants separately. Our results show that BoechRPS18, BoechEfα1, BoechACT2 and BoechUBQ were the most stable genes. Based on geNorm, the combinations of BoechRPS18 and BoechEfα1 or BoechUBQ and BoechEfα1 were the most stable in the apomictic plant, while BoechRPS18 and BoechACT2 or BoechUBQ and BoechACT2 performed best in the sexual plant. When subgroups of tissue samples were analyzed

Validation of suitable reference genes for quantitative gene expression analysis in Panax ginseng

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Meizhen eWang

2016-01-01

Full Text Available Reverse transcription-qPCR (RT-qPCR has become a popular method for gene expression studies. Its results require data normalization by housekeeping genes. No single gene is proved to be stably expressed under all experimental conditions. Therefore, systematic evaluation of reference genes is necessary. With the aim to identify optimum reference genes for RT-qPCR analysis of gene expression in different tissues of Panax ginseng and the seedlings grown under heat stress, we investigated the expression stability of eight candidate reference genes, including elongation factor 1-beta (EF1-β, elongation factor 1-gamma (EF1-γ, eukaryotic translation initiation factor 3G (IF3G, eukaryotic translation initiation factor 3B (IF3B, actin (ACT, actin11 (ACT11, glyceraldehyde-3-phosphate dehydrogenase (GAPDH and cyclophilin ABH-like protein (CYC, using four widely used computational programs: geNorm, Normfinder, BestKeeper, and the comparative ΔCt method. The results were then integrated using the web-based tool RefFinder. As a result, EF1-γ, IF3G and EF1-β were the three most stable genes in different tissues of P. ginseng, while IF3G, ACT11 and GAPDH were the top three-ranked genes in seedlings treated with heat. Using three better reference genes alone or in combination as internal control, we examined the expression profiles of MAR, a multiple function-associated mRNA-like non-coding RNA (mlncRNA in P. ginseng. Taken together, we recommended EF1-γ/IF3G and IF3G/ACT11 as the suitable pair of reference genes for RT-qPCR analysis of gene expression in different tissues of P. ginseng and the seedlings grown under heat stress, respectively. The results serve as a foundation for future studies on P. ginseng functional genomics.

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.

In search of suitable reference genes for gene expression studies of human renal cell carcinoma by real-time PCR

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Kristiansen Glen

2007-06-01

Full Text Available Abstract Background Housekeeping genes are commonly used as endogenous reference genes for the relative quantification of target genes in gene expression studies. No conclusive systematic study comparing the suitability of different candidate reference genes in clear cell renal cell carcinoma has been published to date. To remedy this situation, 10 housekeeping genes for normalizing purposes of RT-PCR measurements already recommended in various studies were examined with regard to their usefulness as reference genes. Results The expression of the potential reference genes was examined in matched malignant and non-malignant tissue specimens from 25 patients with clear cell renal cell carcinoma. Quality assessment of isolated RNA performed with a 2100 Agilent Bioanalyzer showed a mean RNA integrity number of 8.7 for all samples. The between-run variations related to the crossing points of PCR reactions of a control material ranged from 0.17% to 0.38%. The expression of all genes did not depend on age, sex, and tumour stage. Except the genes TATA box binding protein (TBP and peptidylprolyl isomerase A (PPIA, all genes showed significant differences in expression between malignant and non-malignant pairs. The expression stability of the candidate reference genes was additionally controlled using the software programs geNorm and NormFinder. TBP and PPIA were validated as suitable reference genes by normalizing the target gene ADAM9 using these two most stably expressed genes in comparison with up- and down-regulated housekeeping genes of the panel. Conclusion Our study demonstrated the suitability of the two housekeeping genes PPIA and TBP as endogenous reference genes when comparing malignant tissue samples with adjacent normal tissue samples from clear cell renal cell carcinoma. Both genes are recommended as reference genes for relative gene quantification in gene profiling studies either as single gene or preferably in combination.

Assessment of reference gene stability in Rice stripe virus and Rice black streaked dwarf virus infection rice by quantitative Real-time PCR.

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Fang, Peng; Lu, Rongfei; Sun, Feng; Lan, Ying; Shen, Wenbiao; Du, Linlin; Zhou, Yijun; Zhou, Tong

2015-10-24

Stably expressed reference gene(s) normalization is important for the understanding of gene expression patterns by quantitative Real-time PCR (RT-qPCR), particularly for Rice stripe virus (RSV) and Rice black streaked dwarf virus (RBSDV) that caused seriously damage on rice plants in China and Southeast Asia. The expression of fourteen common used reference genes of Oryza sativa L. were evaluated by RT-qPCR in RSV and RBSDV infected rice plants. Suitable normalization reference gene(s) were identified by geNorm and NormFinder algorithms. UBQ 10 + GAPDH and UBC + Actin1 were identified as suitable reference genes for RT-qPCR normalization under RSV and RBSDV infection, respectively. When using multiple reference genes, the expression patterns of OsPRIb and OsWRKY, two virus resistance genes, were approximately similar with that reported previously. Comparatively, by using single reference gene (TIP41-Like), a weaker inducible response was observed. We proposed that the combination of two reference genes could obtain more accurate and reliable normalization of RT-qPCR results in RSV- and RBSDV-infected plants. This work therefore sheds light on establishing a standardized RT-qPCR procedure in RSV- and RBSDV-infected rice plants, and might serve as an important point for discovering complex regulatory networks and identifying genes relevant to biological processes or implicated in virus.

Selection of reliable reference genes in Caenorhabditis elegans for analysis of nanotoxicity.

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Yanqiong Zhang

Full Text Available Despite rapid development and application of a wide range of manufactured metal oxide nanoparticles (NPs, the understanding of potential risks of using NPs is less completed, especially at the molecular level. The nematode Caenorhabditis elegans (C.elegans has been emerging as an environmental model to study the molecular mechanism of environmental contaminations, using standard genetic tools such as the real-time quantitative PCR (RT-qPCR. The most important factor that may affect the accuracy of RT-qPCR is to choose appropriate genes for normalization. In this study, we selected 13 reference gene candidates (act-1, cdc-42, pmp-3, eif-3.C, actin, act-2, csq-1, Y45F10D.4, tba-1, mdh-1, ama-1, F35G12.2, and rbd-1 to test their expression stability under different doses of nano-copper oxide (CuO 0, 1, 10, and 50 µg/mL using RT-qPCR. Four algorithms, geNorm, NormFinder, BestKeeper, and the comparative ΔCt method, were employed to evaluate these 13 candidates expressions. As a result, tba-1, Y45F10D.4 and pmp-3 were the most reliable, which may be used as reference genes in future study of nanoparticle-induced genetic response using C.elegans.

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.

Low-level lasers alter mRNA levels from traditional reference genes used in breast cancer cells

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Teixeira, A. F.; Canuto, K. S.; Rodrigues, J. A.; Fonseca, A. S.; Mencalha, A. L.

2017-07-01

Cancer is among the leading causes of mortality worldwide, increasing the importance of treatment development. Low-level lasers are used in several diseases, but some concerns remains on cancers. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is a technique used to understand cellular behavior through quantification of mRNA levels. Output data from target genes are commonly relative to a reference that cannot vary according to treatment. This study evaluated reference genes levels from MDA-MB-231 cells exposed to red or infrared lasers at different fluences. Cultures were exposed to red and infrared lasers, incubated (4 h, 37 °C), total RNA was extracted and cDNA synthesis was performed to evaluate mRNA levels from ACTB, GUSB and TRFC genes by RT-qPCR. Specific amplification was verified by melting curves and agarose gel electrophoresis. RefFinder enabled data analysis by geNorm, NormFinder and BestKeeper. Specific amplifications were obtained and, although mRNA levels from ACTB, GUSB or TRFC genes presented no significant variation through traditional statistical analysis, Excel-based tools revealed that the use of these reference genes are dependent of laser characteristics. Our data showed that exposure to low-level red and infrared lasers at different fluences alter the mRNA levels from ACTB, GUSB and TRFC in MDA-MB-231 cells.

Evaluation of Reference Genes for Real-Time Quantitative PCR Analysis in Larvae of Spodoptera litura Exposed to Azadirachtin Stress Conditions

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Benshui Shu

2018-04-01

Full Text Available Azadirachtin is an efficient and broad-spectrum botanical insecticide against more than 150 kinds of agricultural pests with the effects of mortality, antifeedant and growth regulation. Real-time quantitative polymerase chain reaction (RT-qPCR could be one of the powerful tools to analyze the gene expression level and investigate the mechanism of azadirachtin at transcriptional level, however, the ideal reference genes are needed to normalize the expression profiling of target genes. In this present study, the fragments of eight candidate reference genes were cloned and identified from the pest Spodoptera litura. In addition, the expression stability of these genes in different samples from larvae of control and azadirachtin treatments were evaluated by the computational methods of NormFinder, BestKeeper, Delta CT, geNorm, and RefFinder. According to our results, two of the reference genes should be the optimal number for RT-qPCR analysis. Furthermore, the best reference genes for different samples were showed as followed: EF-1α and EF2 for cuticle, β-Tubulin and RPL7A for fat body, EF2 and Actin for midgut, EF2 and RPL13A for larva and RPL13A and RPL7A for all the samples. Our results established a reliable normalization for RT-qPCR experiments in S. litura and ensure the data more accurate for the mechanism analysis of azadirachtin.

Evaluation of Reference Genes for Real-Time Quantitative PCR Analysis in Larvae of Spodoptera litura Exposed to Azadirachtin Stress Conditions.

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Shu, Benshui; Zhang, Jingjing; Cui, Gaofeng; Sun, Ranran; Sethuraman, Veeran; Yi, Xin; Zhong, Guohua

2018-01-01

Azadirachtin is an efficient and broad-spectrum botanical insecticide against more than 150 kinds of agricultural pests with the effects of mortality, antifeedant and growth regulation. Real-time quantitative polymerase chain reaction (RT-qPCR) could be one of the powerful tools to analyze the gene expression level and investigate the mechanism of azadirachtin at transcriptional level, however, the ideal reference genes are needed to normalize the expression profiling of target genes. In this present study, the fragments of eight candidate reference genes were cloned and identified from the pest Spodoptera litura . In addition, the expression stability of these genes in different samples from larvae of control and azadirachtin treatments were evaluated by the computational methods of NormFinder, BestKeeper, Delta CT, geNorm, and RefFinder. According to our results, two of the reference genes should be the optimal number for RT-qPCR analysis. Furthermore, the best reference genes for different samples were showed as followed: EF-1α and EF2 for cuticle, β-Tubulin and RPL7A for fat body, EF2 and Actin for midgut, EF2 and RPL13A for larva and RPL13A and RPL7A for all the samples. Our results established a reliable normalization for RT-qPCR experiments in S. litura and ensure the data more accurate for the mechanism analysis of azadirachtin.

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.

Validation of potential reference genes for qPCR in maize across abiotic stresses, hormone treatments, and tissue types.

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Yueai Lin

Full Text Available The reverse transcription quantitative polymerase chain reaction (RT-qPCR is a powerful and widely used technique for the measurement of gene expression. Reference genes, which serve as endogenous controls ensure that the results are accurate and reproducible, are vital for data normalization. To bolster the literature on reference gene selection in maize, ten candidate reference genes, including eight traditionally used internal control genes and two potential candidate genes from our microarray datasets, were evaluated for expression level in maize across abiotic stresses (cold, heat, salinity, and PEG, phytohormone treatments (abscisic acid, salicylic acid, jasmonic acid, ethylene, and gibberellins, and different tissue types. Three analytical software packages, geNorm, NormFinder, and Bestkeeper, were used to assess the stability of reference gene expression. The results revealed that elongation factor 1 alpha (EF1α, tubulin beta (β-TUB, cyclophilin (CYP, and eukaryotic initiation factor 4A (EIF4A were the most reliable reference genes for overall gene expression normalization in maize, while GRP (Glycine-rich RNA-binding protein, GLU1(beta-glucosidase, and UBQ9 (ubiquitin 9 were the least stable and most unsuitable genes. In addition, the suitability of EF1α, β-TUB, and their combination as reference genes was confirmed by validating the expression of WRKY50 in various samples. The current study indicates the appropriate reference genes for the urgent requirement of gene expression normalization in maize across certain abiotic stresses, hormones, and tissue types.

Validation of reference genes in Solenopsis invicta in different developmental stages, castes and tissues.

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Daifeng Cheng

Full Text Available To accurately assess gene expression levels, it is essential to normalize real-time quantitative PCR (RT-qPCR data with suitable internal reference genes. For the red imported fire ant, Solenopsis invicta, reliable reference genes to assess the transcript expression levels of the target genes have not been previously investigated. In this study, we examined the expression levels of five candidate reference genes (rpl18, ef1-beta, act, GAPDH, and tbp in different developmental stages, castes and tissues of S. invicta. To evaluate the suitability of these genes as endogenous controls, three software-based approaches (geNorm, BestKeeper and NormFinder and one web-based comprehensive tool (RefFinder were used to analyze and rank the tested genes. Furthermore, the optimal number of reference gene(s was determined by the pairwise variation value. Our data showed that two of the five candidate genes, rpl18 and ef1-beta, were the most suitable reference genes because they have the most stable expression among different developmental stages, castes and tissues in S. invicta. Although widely used as reference gene in other species, in S. invicta the act gene has high variation in expression and was consequently excluded as a reliable reference gene. The two validated reference genes, rpl18 and ef1-beta, can be widely used for quantification of target gene expression with RT-qPCR technology in S. invicta.

Evaluation of reference gene suitability for quantitative expression analysis by quantitative polymerase chain reaction in the mandibular condyle of sheep.

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Jiang, Xin; Xue, Yang; Zhou, Hongzhi; Li, Shouhong; Zhang, Zongmin; Hou, Rui; Ding, Yuxiang; Hu, Kaijin

2015-10-01

Reference genes are commonly used as a reliable approach to normalize the results of quantitative polymerase chain reaction (qPCR), and to reduce errors in the relative quantification of gene expression. Suitable reference genes belonging to numerous functional classes have been identified for various types of species and tissue. However, little is currently known regarding the most suitable reference genes for bone, specifically for the sheep mandibular condyle. Sheep are important for the study of human bone diseases, particularly for temporomandibular diseases. The present study aimed to identify a set of reference genes suitable for the normalization of qPCR data from the mandibular condyle of sheep. A total of 12 reference genes belonging to various functional classes were selected, and the expression stability of the reference genes was determined in both the normal and fractured area of the sheep mandibular condyle. RefFinder, which integrates the following currently available computational algorithms: geNorm, NormFinder, BestKeeper, and the comparative ΔCt method, was used to compare and rank the candidate reference genes. The results obtained from the four methods demonstrated a similar trend: RPL19, ACTB, and PGK1 were the most stably expressed reference genes in the sheep mandibular condyle. As determined by RefFinder comprehensive analysis, the results of the present study suggested that RPL19 is the most suitable reference gene for studies associated with the sheep mandibular condyle. In addition, ACTB and PGK1 may be considered suitable alternatives.

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.

Selection and validation of reference genes for quantitative gene expression studies in Erythroxylum coca [v1; ref status: indexed, http://f1000r.es/y1

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Teresa Docimo

2013-02-01

Full Text Available Real-time quantitative PCR is a powerful technique for the investigation of comparative gene expression, but its accuracy and reliability depend on the reference genes used as internal standards. Only genes that show a high level of expression stability are suitable for use as reference genes, and these must be identified on a case-by-case basis. Erythroxylum coca produces and accumulates high amounts of the pharmacologically active tropane alkaloid cocaine (especially in the leaves, and is an emerging model for the investigation of tropane alkaloid biosynthesis. The identification of stable internal reference genes for this species is important for its development as a model species, and would enable comparative analysis of candidate biosynthetic genes in the different tissues of the coca plant. In this study, we evaluated the expression stability of nine candidate reference genes in E. coca (Ec6409, Ec10131, Ec11142, Actin, APT2, EF1α, TPB1, Pex4, Pp2aa3. The expression of these genes was measured in seven tissues (flowers, stems, roots and four developmental leaf stages and the stability of expression was assessed using three algorithms (geNorm, NormFinder and BestKeeper. From our results we conclude that Ec10131 and TPB1 are the most appropriate internal reference genes in leaves (where the majority of cocaine is produced, while Ec10131 and Ec6409 are the most suitable internal reference genes across all of the tissues tested.

Suitable Reference Genes for Accurate Gene Expression Analysis in Parsley (Petroselinum crispum) for Abiotic Stresses and Hormone Stimuli.

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Li, Meng-Yao; Song, Xiong; Wang, Feng; Xiong, Ai-Sheng

2016-01-01

Parsley, one of the most important vegetables in the Apiaceae family, is widely used in the food, medicinal, and cosmetic industries. Recent studies on parsley mainly focus on its chemical composition, and further research involving the analysis of the plant's gene functions and expressions is required. qPCR is a powerful method for detecting very low quantities of target transcript levels and is widely used to study gene expression. To ensure the accuracy of results, a suitable reference gene is necessary for expression normalization. In this study, four software, namely geNorm, NormFinder, BestKeeper, and RefFinder were used to evaluate the expression stabilities of eight candidate reference genes of parsley ( GAPDH, ACTIN, eIF-4 α, SAND, UBC, TIP41, EF-1 α, and TUB ) under various conditions, including abiotic stresses (heat, cold, salt, and drought) and hormone stimuli treatments (GA, SA, MeJA, and ABA). Results showed that EF-1 α and TUB were the most stable genes for abiotic stresses, whereas EF-1 α, GAPDH , and TUB were the top three choices for hormone stimuli treatments. Moreover, EF-1 α and TUB were the most stable reference genes among all tested samples, and UBC was the least stable one. Expression analysis of PcDREB1 and PcDREB2 further verified that the selected stable reference genes were suitable for gene expression normalization. This study can guide the selection of suitable reference genes in gene expression in parsley.

Suitable reference genes for accurate gene expression analysis in parsley (Petroselinum crispum for abiotic stresses and hormone stimuli

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Meng-Yao Li

2016-09-01

Full Text Available Parsley is one of the most important vegetable in Apiaceae family and widely used in food industry, medicinal and cosmetic. The recent studies in parsley are mainly focus on chemical composition, further research involving the analysis of the gene functions and expressions will be required. qPCR is a powerful method for detecting very low quantities of target transcript levels and widely used for gene expression studies. To ensure the accuracy of results, a suitable reference gene is necessary for expression normalization. In this study, three software geNorm, NormFinder, and BestKeeper were used to evaluate the expression stabilities of eight candidate reference genes (GAPDH, ACTIN, eIF-4α, SAND, UBC, TIP41, EF-1α, and TUB under various conditions including abiotic stresses (heat, cold, salt, and drought and hormone stimuli treatments (GA, SA, MeJA, and ABA. The results showed that EF-1α and TUB were identified as the most stable genes for abiotic stresses, while EF-1α, GAPDH, and TUB were the top three choices for hormone stimuli treatments. Moreover, EF-1α and TUB were the most stable reference genes across all the tested samples, while UBC was the least stable one. The expression analysis of PcDREB1 and PcDREB2 further verified that the selected stable reference genes were suitable for gene expression normalization. This study provides a guideline for selection the suitable reference genes in gene expression in parsley.

Selection of housekeeping genes and demonstration of RNAi in cotton leafhopper, Amrasca biguttula biguttula (Ishida)

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Gupta, Mridula; Pandher, Suneet; Kaur, Gurmeet; Rathore, Pankaj; Palli, Subba Reddy

2018-01-01

Amrasca biguttula biguttula (Ishida) commonly known as cotton leafhopper is a severe pest of cotton and okra. Not much is known on this insect at molecular level due to lack of genomic and transcriptomic data. To prepare for functional genomic studies in this insect, we evaluated 15 common housekeeping genes (Tub, B-Tub, EF alpha, GADPH, UbiCF, RP13, Ubiq, G3PD, VATPase, Actin, 18s, 28s, TATA, ETF, SOD and Cytolytic actin) during different developmental stages and under starvation stress. We selected early (1st and 2nd), late (3rd and 4th) stage nymphs and adults for identification of stable housekeeping genes using geNorm, NormFinder, BestKeeper and RefFinder software. Based on the different algorithms, RP13 and VATPase are identified as the most suitable reference genes for quantification of gene expression by reverse transcriptase quantitative PCR (RT-qPCR). Based on RefFinder which comprehended the results of three algorithms, RP13 in adults, Tubulin (Tub) in late nymphs, 28S in early nymph and UbiCF under starvation stress were identified as the most stable genes. We also developed methods for feeding double-stranded RNA (dsRNA) incorporated in the diet. Feeding dsRNA targeting Snf7, IAP, AQP1, and VATPase caused 56.17–77.12% knockdown of targeted genes compared to control and 16 to 48% mortality of treated insects when compared to control. PMID:29329327

Selection of housekeeping genes and demonstration of RNAi in cotton leafhopper, Amrasca biguttula biguttula (Ishida.

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Satnam Singh

Full Text Available Amrasca biguttula biguttula (Ishida commonly known as cotton leafhopper is a severe pest of cotton and okra. Not much is known on this insect at molecular level due to lack of genomic and transcriptomic data. To prepare for functional genomic studies in this insect, we evaluated 15 common housekeeping genes (Tub, B-Tub, EF alpha, GADPH, UbiCF, RP13, Ubiq, G3PD, VATPase, Actin, 18s, 28s, TATA, ETF, SOD and Cytolytic actin during different developmental stages and under starvation stress. We selected early (1st and 2nd, late (3rd and 4th stage nymphs and adults for identification of stable housekeeping genes using geNorm, NormFinder, BestKeeper and RefFinder software. Based on the different algorithms, RP13 and VATPase are identified as the most suitable reference genes for quantification of gene expression by reverse transcriptase quantitative PCR (RT-qPCR. Based on RefFinder which comprehended the results of three algorithms, RP13 in adults, Tubulin (Tub in late nymphs, 28S in early nymph and UbiCF under starvation stress were identified as the most stable genes. We also developed methods for feeding double-stranded RNA (dsRNA incorporated in the diet. Feeding dsRNA targeting Snf7, IAP, AQP1, and VATPase caused 56.17-77.12% knockdown of targeted genes compared to control and 16 to 48% mortality of treated insects when compared to control.

Identification of reference genes for relative quantification of circulating microRNAs in bovine serum.

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In-Seon Bae

Full Text Available Circulating microRNAs in body fluids have been implicated as promising biomarkers for physiopathology disorders. Currently, the expression levels of circulating microRNAs are estimated by reverse transcription quantitative real-time polymerase chain reaction. Use of appropriate reference microRNAs for normalization is critical for accurate microRNA expression analysis. However, no study has systematically investigated reference genes for evaluating circulating microRNA expression in cattle. In this study, we describe the identification and characterization of appropriate reference microRNAs for use in the normalization of circulating microRNA levels in bovine serum. We evaluated the expression stability of ten candidate reference genes in bovine serum by using reverse transcription quantitative real-time polymerase chain reaction. Data were analyzed using geNorm, NormFinder, and BestKeeper statistical algorithms. The results consistently showed that a combination of miR-93 and miR-127 provided the most stably expressed reference. The suitability of these microRNAs was validated, and even when compared among different genders or breeds, the combination of miR-93 and miR-127 was ranked as the most stable microRNA reference. Therefore, we conclude that this combination is the optimal endogenous reference for reverse transcription quantitative real-time polymerase chain reaction-based detection of microRNAs in bovine serum. The data presented in this study are crucial to successful biomarker discovery and validation for the diagnosis of physiopathological conditions in cattle.

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.

Evaluation of Appropriate Reference Genes for Gene Expression Normalization during Watermelon Fruit Development.

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Qiusheng Kong

Full Text Available Gene expression analysis in watermelon (Citrullus lanatus fruit has drawn considerable attention with the availability of genome sequences to understand the regulatory mechanism of fruit development and to improve its quality. Real-time quantitative reverse-transcription PCR (qRT-PCR is a routine technique for gene expression analysis. However, appropriate reference genes for transcript normalization in watermelon fruits have not been well characterized. The aim of this study was to evaluate the appropriateness of 12 genes for their potential use as reference genes in watermelon fruits. Expression variations of these genes were measured in 48 samples obtained from 12 successive developmental stages of parthenocarpic and fertilized fruits of two watermelon genotypes by using qRT-PCR analysis. Considering the effects of genotype, fruit setting method, and developmental stage, geNorm determined clathrin adaptor complex subunit (ClCAC, β-actin (ClACT, and alpha tubulin 5 (ClTUA5 as the multiple reference genes in watermelon fruit. Furthermore, ClCAC alone or together with SAND family protein (ClSAND was ranked as the single or two best reference genes by NormFinder. By using the top-ranked reference genes to normalize the transcript abundance of phytoene synthase (ClPSY1, a good correlation between lycopene accumulation and ClPSY1 expression pattern was observed in ripening watermelon fruit. These validated reference genes will facilitate the accurate measurement of gene expression in the studies on watermelon fruit biology.

Identification of Reference Genes for Quantitative Real Time PCR Assays in Aortic Tissue of Syrian Hamsters with Bicuspid Aortic Valve.

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Rueda-Martínez, Carmen; Fernández, M Carmen; Soto-Navarrete, María Teresa; Jiménez-Navarro, Manuel; Durán, Ana Carmen; Fernández, Borja

2016-01-01

Bicuspid aortic valve (BAV) is the most frequent congenital cardiac malformation in humans, and appears frequently associated with dilatation of the ascending aorta. This association is likely the result of a common aetiology. Currently, a Syrian hamster strain with a relatively high (∼40%) incidence of BAV constitutes the only spontaneous animal model of BAV disease. The characterization of molecular alterations in the aorta of hamsters with BAV may serve to identify pathophysiological mechanisms and molecular markers of disease in humans. In this report, we evaluate the expression of ten candidate reference genes in aortic tissue of hamsters in order to identify housekeeping genes for normalization using quantitative real time PCR (RT-qPCR) assays. A total of 51 adult (180-240 days old) and 56 old (300-440 days old) animals were used. They belonged to a control strain of hamsters with normal, tricuspid aortic valve (TAV; n = 30), or to the affected strain of hamsters with TAV (n = 45) or BAV (n = 32). The expression stability of the candidate reference genes was determined by RT-qPCR using three statistical algorithms, GeNorm, NormFinder and Bestkeeper. The expression analyses showed that the most stable reference genes for the three algorithms employed were Cdkn1β, G3pdh and Polr2a. We propose the use of Cdkn1β, or both Cdkn1β and G3pdh as reference genes for mRNA expression analyses in Syrian hamster aorta.

Identification of Reference Genes for Quantitative Real Time PCR Assays in Aortic Tissue of Syrian Hamsters with Bicuspid Aortic Valve.

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Carmen Rueda-Martínez

Full Text Available Bicuspid aortic valve (BAV is the most frequent congenital cardiac malformation in humans, and appears frequently associated with dilatation of the ascending aorta. This association is likely the result of a common aetiology. Currently, a Syrian hamster strain with a relatively high (∼40% incidence of BAV constitutes the only spontaneous animal model of BAV disease. The characterization of molecular alterations in the aorta of hamsters with BAV may serve to identify pathophysiological mechanisms and molecular markers of disease in humans. In this report, we evaluate the expression of ten candidate reference genes in aortic tissue of hamsters in order to identify housekeeping genes for normalization using quantitative real time PCR (RT-qPCR assays. A total of 51 adult (180-240 days old and 56 old (300-440 days old animals were used. They belonged to a control strain of hamsters with normal, tricuspid aortic valve (TAV; n = 30, or to the affected strain of hamsters with TAV (n = 45 or BAV (n = 32. The expression stability of the candidate reference genes was determined by RT-qPCR using three statistical algorithms, GeNorm, NormFinder and Bestkeeper. The expression analyses showed that the most stable reference genes for the three algorithms employed were Cdkn1β, G3pdh and Polr2a. We propose the use of Cdkn1β, or both Cdkn1β and G3pdh as reference genes for mRNA expression analyses in Syrian hamster aorta.

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.

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.

Comprehensive evaluation of candidate reference genes for gene expression studies in Lysiphlebia japonica (Hymenoptera: Aphidiidae) using RT-qPCR.

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Gao, Xue-Ke; Zhang, Shuai; Luo, Jun-Yu; Wang, Chun-Yi; Lü, Li-Min; Zhang, Li-Juan; Zhu, Xiang-Zhen; Wang, Li; Lu, Hui; Cui, Jin-Jie

2017-12-30

Lysiphlebia japonica (Ashmead) is a predominant parasitoid of cotton-melon aphids in the fields of northern China with a proven ability to effectively control cotton aphid populations in early summer. For accurate normalization of gene expression in L. japonica using quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR), reference genes with stable gene expression patterns are essential. However, no appropriate reference genes is L. japonica have been investigated to date. In the present study, 12 selected housekeeping genes from L. japonica were cloned. We evaluated the stability of these genes under various experimental treatments by RT-qPCR using four independent (geNorm, NormFinder, BestKeeper and Delta Ct) and one comparative (RefFinder) algorithm. We identified genes showing the most stable levels of expression: DIMT, 18S rRNA, and RPL13 during different stages; AK, RPL13, and TBP among sexes; EF1A, PPI, and RPL27 in different tissues, and EF1A, RPL13, and PPI in adults fed on different diets. Moreover, the expression profile of a target gene (odorant receptor 1, OR1) studied during the developmental stages confirms the reliability of the chosen selected reference genes. This study provides for the first time a comprehensive list of suitable reference genes for gene expression studies in L. japonica and will benefit subsequent genomics and functional genomics research on this natural enemy. Copyright © 2017. Published by Elsevier B.V.

Identification of Reliable Reference Genes for Quantification of MicroRNAs in Serum Samples of Sulfur Mustard-Exposed Veterans.

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Gharbi, Sedigheh; Shamsara, Mehdi; Khateri, Shahriar; Soroush, Mohammad Reza; Ghorbanmehr, Nassim; Tavallaei, Mahmood; Nourani, Mohammad Reza; Mowla, Seyed Javad

2015-01-01

In spite of accumulating information about pathological aspects of sulfur mustard (SM), the precise mechanism responsible for its effects is not well understood. Circulating microRNAs (miRNAs) are promising biomarkers for disease diagnosis and prognosis. Accurate normalization using appropriate reference genes, is a critical step in miRNA expression studies. In this study, we aimed to identify appropriate reference gene for microRNA quantification in serum samples of SM victims. In this case and control experimental study, using quantitative real-time polymerase chain reaction (qRT-PCR), we evaluated the suitability of a panel of small RNAs including SNORD38B, SNORD49A, U6, 5S rRNA, miR-423-3p, miR-191, miR-16 and miR-103 in sera of 28 SM-exposed veterans of Iran-Iraq war (1980-1988) and 15 matched control volunteers. Different statistical algorithms including geNorm, Normfinder, best-keeper and comparative delta-quantification cycle (Cq) method were employed to find the least variable reference gene. miR-423-3p was identified as the most stably expressed reference gene, and miR- 103 and miR-16 ranked after that. We demonstrate that non-miRNA reference genes have the least stabil- ity in serum samples and that some house-keeping miRNAs may be used as more reliable reference genes for miRNAs in serum. In addition, using the geometric mean of two reference genes could increase the reliability of the normalizers.

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.

Evaluation of Appropriate Reference Genes for Gene Expression Normalization during Watermelon Fruit Development.

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Kong, Qiusheng; Yuan, Jingxian; Gao, Lingyun; Zhao, Liqiang; Cheng, Fei; Huang, Yuan; Bie, Zhilong

2015-01-01

Gene expression analysis in watermelon (Citrullus lanatus) fruit has drawn considerable attention with the availability of genome sequences to understand the regulatory mechanism of fruit development and to improve its quality. Real-time quantitative reverse-transcription PCR (qRT-PCR) is a routine technique for gene expression analysis. However, appropriate reference genes for transcript normalization in watermelon fruits have not been well characterized. The aim of this study was to evaluate the appropriateness of 12 genes for their potential use as reference genes in watermelon fruits. Expression variations of these genes were measured in 48 samples obtained from 12 successive developmental stages of parthenocarpic and fertilized fruits of two watermelon genotypes by using qRT-PCR analysis. Considering the effects of genotype, fruit setting method, and developmental stage, geNorm determined clathrin adaptor complex subunit (ClCAC), β-actin (ClACT), and alpha tubulin 5 (ClTUA5) as the multiple reference genes in watermelon fruit. Furthermore, ClCAC alone or together with SAND family protein (ClSAND) was ranked as the single or two best reference genes by NormFinder. By using the top-ranked reference genes to normalize the transcript abundance of phytoene synthase (ClPSY1), a good correlation between lycopene accumulation and ClPSY1 expression pattern was observed in ripening watermelon fruit. These validated reference genes will facilitate the accurate measurement of gene expression in the studies on watermelon fruit biology.

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.

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

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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 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.

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

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

Validation of reference genes for quantitative RT-PCR normalization in Suaeda aralocaspica, an annual halophyte with heteromorphism and C4 pathway without Kranz anatomy

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Jing Cao

2016-02-01

Full Text Available Reverse transcription quantitative real-time polymerase chain reaction (qRT-PCR is a powerful analytical technique for the measurement of gene expression, which depends on the stability of the reference gene used for data normalization. Suaeda aralocaspica, an annual halophyte with heteromorphic seeds and possessing C4 photosynthesis pathway without Kranz anatomy, is an ideal plant species to identify stress tolerance-related genes and compare relative expression at transcriptional level. So far, no molecular information is available for this species. In the present study, six traditionally used reference genes were selected and their expression stability in two types of seeds of S. aralocaspica under different experimental conditions was evaluated. Three analytical programs, geNorm, NormFinder and BestKeeper, were used to assess and rank the stability of reference gene expression. Results revealed that although some reference genes may display different transcriptional profiles between the two types of seeds, β-TUB and GAPDH appeared to be the most suitable references under different developmental stages and tissues. GAPDH was the appropriate reference gene under different germination time points and salt stress conditions, and ACTIN was suitable for various abiotic stress treatments for the two types of seeds. For all the sample pools, β-TUB served as the most stable reference gene, whereas 18S rRNA and 28S rRNA performed poorly and presented as the least stable genes in our study. UBQ seemed to be unsuitable as internal control under different salt treatments. In addition, the expression of a photosynthesis-related gene (PPDK of C4 pathway and a salt tolerance-related gene (SAT of S. aralocaspica were used to validate the best performance reference genes. This is the first systematic comparison of reference gene selection for qRT-PCR work in S. aralocaspica and these data will facilitate further studies on gene expression in this species

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

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

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

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 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 q

Exploring valid reference genes for quantitative real - time rt - pce studies of hydrogenperoxide signaling in arabidopsis

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Zhou, H.; Han, B.; Xie, Y.; Zhang, J.; Shen, W.

2015-01-01

Hydrogen peroxide (H/sub 2/O/sub 2/ ) acts as a signaling molecule modulating the expression of various genes in plants. However, the reference gene(s) used for gene expression analysis of H/sub 2/O/sub 2/ signaling is still arbitrary. A reliable result obtained by quantitative real-time RT-PCR (RT-qPCR) highly depends on accurate transcript normalization using stably expressed reference genes, whereas the inaccurate normalization could easily lead to the false conclusions. In this report, by using geNorm and NormFinder algorithms, 12 candidate reference genes were evaluated and compared in root and shoot tissues of Arabidopsis upon different doses of H/sub 2/O/sub 2/. The results revealed that, in our experimental conditions, three novel reference genes (TIP41-like, UKN, and UBC21) were identified and validated as suitable reference genes for RT-qPCR normalization in both root and shoot tissues under oxidative stress. This conclusion was further confirmed by publicly available microarray data of methyl viologen and drought stress. In comparison with a single reference gene (EF-1a), the expression pattern of ZAT12 modulated by H/sub 2/O/sub 2/, when using TIP41-like, UKN, and UBC21 as multiple reference gene(s), was similar with the previous reports by using northern blotting. Thus, we proposed that these three reference genes might be good candidates for other researchers to include in their reference gene validation in gene expression studies under H/sub 2/O/sub 2/ related oxidative stress. (author)

Saponin determination, expression analysis and functional characterization of saponin biosynthetic genes in Chenopodium quinoa leaves.

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Fiallos-Jurado, Jennifer; Pollier, Jacob; Moses, Tessa; Arendt, Philipp; Barriga-Medina, Noelia; Morillo, Eduardo; Arahana, Venancio; de Lourdes Torres, Maria; Goossens, Alain; Leon-Reyes, Antonio

2016-09-01

Quinoa (Chenopodium quinoa Willd.) is a highly nutritious pseudocereal with an outstanding protein, vitamin, mineral and nutraceutical content. The leaves, flowers and seed coat of quinoa contain triterpenoid saponins, which impart bitterness to the grain and make them unpalatable without postharvest removal of the saponins. In this study, we quantified saponin content in quinoa leaves from Ecuadorian sweet and bitter genotypes and assessed the expression of saponin biosynthetic genes in leaf samples elicited with methyl jasmonate. We found saponin accumulation in leaves after MeJA treatment in both ecotypes tested. As no reference genes were available to perform qPCR in quinoa, we mined publicly available RNA-Seq data for orthologs of 22 genes known to be stably expressed in Arabidopsis thaliana using geNorm, NormFinder and BestKeeper algorithms. The quinoa ortholog of At2g28390 (Monensin Sensitivity 1, MON1) was stably expressed and chosen as a suitable reference gene for qPCR analysis. Candidate saponin biosynthesis genes were screened in the quinoa RNA-Seq data and subsequent functional characterization in yeast led to the identification of CqbAS1, CqCYP716A78 and CqCYP716A79. These genes were found to be induced by MeJA, suggesting this phytohormone might also modulate saponin biosynthesis in quinoa leaves. Knowledge of the saponin biosynthesis and its regulation in quinoa may aid the further development of sweet cultivars that do not require postharvest processing. Copyright © 2016 Elsevier Ireland Ltd. 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 gene expression studies of human stomach cancer by reverse transcription-qPCR

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Lee Yeon-Su

2010-05-01

Full Text Available Abstract Background Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR is a powerful method for the analysis of gene expression. Target gene expression levels are usually normalized to a consistently expressed reference gene also known as internal standard, in the same sample. However, much effort has not been expended thus far in the search for reference genes suitable for the study of stomach cancer using RT-qPCR, although selection of optimal reference genes is critical for interpretation of results. Methods We assessed the suitability of six possible reference genes, beta-actin (ACTB, glyceraldehydes-3-phosphate dehydrogenase (GAPDH, hypoxanthine phosphoribosyl transferase 1 (HPRT1, beta-2-microglobulin (B2M, ribosomal subunit L29 (RPL29 and 18S ribosomal RNA (18S rRNA in 20 normal and tumor stomach tissue pairs of stomach cancer patients and 6 stomach cancer cell lines, by RT-qPCR. Employing expression stability analyses using NormFinder and geNorm algorithms we determined the order of performance of these reference genes and their variation values. Results This RT-qPCR study showed that there are statistically significant (p Conclusion This study validated RPL29 and RPL29-B2M as the best single reference genes and combination, for RT-qPCR analysis of 'all stomach tissues', and B2M and B2M-GAPDH as the best single reference gene and combination, for 'stomach cancer cell lines'. Use of these validated reference genes should provide more exact interpretation of differential gene expressions at transcription level in stomach cancer.

Optimal Reference Genes for Gene Expression Normalization in Trichomonas vaginalis

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dos Santos, Odelta; de Vargas Rigo, Graziela; Frasson, Amanda Piccoli; Macedo, Alexandre José; Tasca, Tiana

2015-01-01

Trichomonas vaginalis is the etiologic agent of trichomonosis, the most common non-viral sexually transmitted disease worldwide. This infection is associated with several health consequences, including cervical and prostate cancers and HIV acquisition. Gene expression analysis has been facilitated because of available genome sequences and large-scale transcriptomes in T. vaginalis, particularly using quantitative real-time polymerase chain reaction (qRT-PCR), one of the most used methods for molecular studies. Reference genes for normalization are crucial to ensure the accuracy of this method. However, to the best of our knowledge, a systematic validation of reference genes has not been performed for T. vaginalis. In this study, the transcripts of nine candidate reference genes were quantified using qRT-PCR under different cultivation conditions, and the stability of these genes was compared using the geNorm and NormFinder algorithms. The most stable reference genes were α-tubulin, actin and DNATopII, and, conversely, the widely used T. vaginalis reference genes GAPDH and β-tubulin were less stable. The PFOR gene was used to validate the reliability of the use of these candidate reference genes. As expected, the PFOR gene was upregulated when the trophozoites were cultivated with ferrous ammonium sulfate when the DNATopII, α-tubulin and actin genes were used as normalizing gene. By contrast, the PFOR gene was downregulated when the GAPDH gene was used as an internal control, leading to misinterpretation of the data. These results provide an important starting point for reference gene selection and gene expression analysis with qRT-PCR studies of T. vaginalis. PMID:26393928

Selection and validation of reference genes for quantitative gene expression analyses in various tissues and seeds at different developmental stages in Bixa orellana L.

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Moreira, Viviane S; Soares, Virgínia L F; Silva, Raner J S; Sousa, Aurizangela O; Otoni, Wagner C; Costa, Marcio G C

2018-05-01

Bixa orellana L., popularly known as annatto, produces several secondary metabolites of pharmaceutical and industrial interest, including bixin, whose molecular basis of biosynthesis remain to be determined. Gene expression analysis by quantitative real-time PCR (qPCR) is an important tool to advance such knowledge. However, correct interpretation of qPCR data requires the use of suitable reference genes in order to reduce experimental variations. In the present study, we have selected four different candidates for reference genes in B. orellana , coding for 40S ribosomal protein S9 (RPS9), histone H4 (H4), 60S ribosomal protein L38 (RPL38) and 18S ribosomal RNA (18SrRNA). Their expression stabilities in different tissues (e.g. flower buds, flowers, leaves and seeds at different developmental stages) were analyzed using five statistical tools (NormFinder, geNorm, BestKeeper, ΔCt method and RefFinder). The results indicated that RPL38 is the most stable gene in different tissues and stages of seed development and 18SrRNA is the most unstable among the analyzed genes. In order to validate the candidate reference genes, we have analyzed the relative expression of a target gene coding for carotenoid cleavage dioxygenase 1 (CCD1) using the stable RPL38 and the least stable gene, 18SrRNA , for normalization of the qPCR data. The results demonstrated significant differences in the interpretation of the CCD1 gene expression data, depending on the reference gene used, reinforcing the importance of the correct selection of reference genes for normalization.

Identification of Importin 8 (IPO8 as the most accurate reference gene for the clinicopathological analysis of lung specimens

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Pio Ruben

2008-11-01

Full Text Available Abstract Background The accurate normalization of differentially expressed genes in lung cancer is essential for the identification of novel therapeutic targets and biomarkers by real time RT-PCR and microarrays. Although classical "housekeeping" genes, such as GAPDH, HPRT1, and beta-actin have been widely used in the past, their accuracy as reference genes for lung tissues has not been proven. Results We have conducted a thorough analysis of a panel of 16 candidate reference genes for lung specimens and lung cell lines. Gene expression was measured by quantitative real time RT-PCR and expression stability was analyzed with the softwares GeNorm and NormFinder, mean of |ΔCt| (= |Ct Normal-Ct tumor| ± SEM, and correlation coefficients among genes. Systematic comparison between candidates led us to the identification of a subset of suitable reference genes for clinical samples: IPO8, ACTB, POLR2A, 18S, and PPIA. Further analysis showed that IPO8 had a very low mean of |ΔCt| (0.70 ± 0.09, with no statistically significant differences between normal and malignant samples and with excellent expression stability. Conclusion Our data show that IPO8 is the most accurate reference gene for clinical lung specimens. In addition, we demonstrate that the commonly used genes GAPDH and HPRT1 are inappropriate to normalize data derived from lung biopsies, although they are suitable as reference genes for lung cell lines. We thus propose IPO8 as a novel reference gene for lung cancer samples.

Identifying optimal reference genes for the normalization of microRNA expression in cucumber under viral stress

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Liang, Chaoqiong; Hao, Jianjun; Meng, Yan; Luo, Laixin; Li, Jianqiang

2018-01-01

Cucumber green mottle mosaic virus (CGMMV) is an economically important pathogen and causes significant reduction of both yield and quality of cucumber (Cucumis sativus). Currently, there were no satisfied strategies for controlling the disease. A better understanding of microRNA (miRNA) expression related to the regulation of plant-virus interactions and virus resistance would be of great assistance when developing control strategies for CGMMV. However, accurate expression analysis is highly dependent on robust and reliable reference gene used as an internal control for normalization of miRNA expression. Most commonly used reference genes involved in CGMMV-infected cucumber are not universally expressed depending on tissue types and stages of plant development. It is therefore crucial to identify suitable reference genes in investigating the role of miRNA expression. In this study, seven reference genes, including Actin, Tubulin, EF-1α, 18S rRNA, Ubiquitin, GAPDH and Cyclophilin, were evaluated for the most accurate results in analyses using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Gene expression was assayed on cucumber leaves, stems and roots that were collected at different days post inoculation with CGMMV. The expression data were analyzed using algorithms including delta-Ct, geNorm, NormFinder, and BestKeeper as well as the comparative tool RefFinder. The reference genes were subsequently validated using miR159. The results showed that EF-1α and GAPDH were the most reliable reference genes for normalizing miRNA expression in leaf, root and stem samples, while Ubiquitin and EF-1α were the most suitable combination overall. PMID:29543906

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.

Reference Genes for qPCR Analysis in Resin-Tapped Adult Slash Pine As a Tool to Address the Molecular Basis of Commercial Resinosis

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Júlio C. de Lima

2016-06-01

Full Text Available Pine oleoresin is a major source of terpenes, consisting of turpentine (mono- and sesquiterpenes and rosin (diterpenes fractions. Higher oleoresin yields are of economic interest, since oleoresin derivatives make up a valuable source of materials for chemical industries. Oleoresin can be extracted from living trees, often by the bark streak method, in which bark removal is done periodically, followed by application of stimulant paste containing sulfuric acid and other chemicals on the freshly wounded exposed surface. To better understand the molecular basis of chemically-stimulated and wound induced oleoresin production, we evaluated the stability of 11 putative reference genes for the purpose of normalization in studying Pinus elliottii gene expression during oleoresinosis. Samples for RNA extraction were collected from field-grown adult trees under tapping operations using stimulant pastes with different compositions and at various time points after paste application. Statistical methods established by geNorm, NormFinder, and BestKeeper softwares were consistent in pointing as adequate reference genes HISTO3 and UBI. To confirm expression stability of the candidate reference genes, expression profiles of putative P. elliottii orthologs of resin biosynthesis-related genes encoding Pinus contorta β-pinene synthase [PcTPS-(−β-pin1], P. contorta levopimaradiene/abietadiene synthase (PcLAS1, Pinus taeda α-pinene synthase [PtTPS-(+αpin], and P. taeda α-farnesene synthase (PtαFS were examined following stimulant paste application. Increased oleoresin yields observed in stimulated treatments using phytohormone-based pastes were consistent with higher expression of pinene synthases. Overall, the expression of all genes examined matched the expected profiles of oleoresin-related transcript changes reported for previously examined conifers.

Housekeeping genes for quantitative expression studies in the three-spined stickleback Gasterosteus aculeatus

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Becker Sven

2008-01-01

Full Text Available Abstract Background During the last years the quantification of immune response under immunological challenges, e.g. parasitation, has been a major focus of research. In this context, the expression of immune response genes in teleost fish has been surveyed for scientific and commercial purposes. Despite the fact that it was shown in teleostei and other taxa that the gene for beta-actin is not the most stably expressed housekeeping gene (HKG, depending on the tissue and experimental treatment, the gene has been used as a reference gene in such studies. In the three-spined stickleback, Gasterosteus aculeatus, other HKG than the one for beta-actin have not been established so far. Results To establish a reliable method for the measurement of immune gene expression in Gasterosteus aculeatus, sequences from the now available genome database and an EST library of the same species were used to select oligonucleotide primers for HKG, in order to perform quantitative reverse-transcription (RT PCR. The expression stability of ten candidate reference genes was evaluated in three different tissues, and in five parasite treatment groups, using the three algorithms BestKeeper, geNorm and NormFinder. Our results showed that in most of the tissues and treatments HKG that could not be used so far due to unknown sequences, proved to be more stably expressed than the one for beta-actin. Conclusion As they were the most stably expressed genes in all tissues examined, we suggest using the genes for the L13a ribosomal binding protein and ubiquitin as alternative or additional reference genes in expression analysis in Gasterosteus aculeatus.

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.

Evaluating the potential of housekeeping genes, rRNAs, snRNAs, microRNAs and circRNAs as reference genes for the estimation of PMI.

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Tu, Chunyan; Du, Tieshuai; Shao, Chengchen; Liu, Zengjia; Li, Liliang; Shen, Yiwen

2018-04-24

The precise estimation of postmortem interval (PMI) is a critical step in death investigation of forensic cases. Detecting the degradation of RNA in tissues by real time quantitative polymerase chain reaction (RT-qPCR) technology provides a new theoretical basis for estimation of PMI. However, most commonly used reference genes degrade over time, while previous studies seldom consider this when selecting suitable reference genes for the estimation of PMI. Studies have shown microRNAs (miRNAs) are very stable and circular RNAs (circRNAs) have recently emerged as a novel class of RNAs with high stability. We aimed to evaluate the stability of the two kinds of RNAs and normal reference genes using geNorm and NormFinder algorithms to identify tissue-specific reference genes for PMI estimation. The content of candidate RNAs from mouse heart, liver and skeletal muscle tissues were dynamically examined in 8 consecutive days after death. Among the 11 candidate genes (β-actin, Gapdh, Rps18, 5S, 18S, U6, miR-133a, miR-122, circ-AFF1, LC-Ogdh and LC-LRP6), the following genes showed prioritized stability: miR-122, miR-133a and 18S in heart tissues; LC-Ogdh, circ-AFF1 and miR-122 in liver tissues; and miR-133a, circ-AFF1 and LC-LRP6 in skeletal muscle tissues. Our results suggested that miRNAs and circRNAs were more stable as reference genes than other kinds of RNAs regarding PMI estimation. The appropriate internal control genes were not completely the same across tissue types.

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.

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

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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.

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.

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.

Validation of suitable reference genes for expression studies in different pilocarpine-induced models of mesial temporal lobe epilepsy.

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Thalita Ewellyn Batista Sales Marques

Full Text Available It is well recognized that the reference gene in a RT-qPCR should be properly validated to ensure that gene expression is unaffected by the experimental condition. We investigated eight potential reference genes in two different pilocarpine PILO-models of mesial temporal lobe epilepsy (MTLE performing a stability expression analysis using geNorm, NormFinder and BestKepeer softwares. Then, as a validation strategy, we conducted a relative expression analysis of the Gfap gene. Our results indicate that in the systemic PILO-model Actb, Gapdh, Rplp1, Tubb2a and Polr1a mRNAs were highly stable in hippocampus of rats from all experimental and control groups, whereas Gusb revealed to be the most variable one. In fact, we observed that using Gusb for normalization, the relative mRNA levels of the Gfap gene differed from those obtained with stable genes. On the contrary, in the intrahippocampal PILO-model, all softwares included Gusb as a stable gene, whereas B2m was indicated as the worst candidate gene. The results obtained for the other reference genes were comparable to those observed for the systemic Pilo-model. The validation of these data by the analysis of the relative expression of Gfap showed that the upregulation of the Gfap gene in the hippocampus of rats sacrificed 24 hours after status epilepticus (SE was undetected only when B2m was used as the normalizer. These findings emphasize that a gene that is stable in one pathology model may not be stable in a different experimental condition related to the same pathology and therefore, the choice of reference genes depends on study design.

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

Selection and validation of appropriate reference genes for quantitative real-time PCR analysis in Salvia hispanica.

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Rahul Gopalam

Full Text Available Quantitative real-time polymerase chain reaction (qRT-PCR has become the most popular choice for gene expression studies. For accurate expression analysis, it is pertinent to select a stable reference gene to normalize the data. It is now known that the expression of internal reference genes varies considerably during developmental stages and under different experimental conditions. For Salvia hispanica, an economically important oilseed crop, there are no reports of stable reference genes till date. In this study, we chose 13 candidate reference genes viz. Actin11 (ACT, Elongation factor 1-alpha (EF1-α, Eukaryotic translation initiation factor 3E (ETIF3E, alpha tubulin (α-TUB, beta tubulin (β-TUB, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Cyclophilin (CYP, Clathrin adaptor complex (CAC, Serine/threonine-protein phosphatase 2A (PP2A, FtsH protease (FtsH, 18S ribosomal RNA (18S rRNA, S-adenosyl methionine decarboxylase (SAMDC and Rubisco activase (RCA and the expression levels of these genes were assessed in a diverse set of tissue samples representing vegetative stages, reproductive stages and various abiotic stress treatments. Two of the widely used softwares, geNorm and Normfinder were used to evaluate the expression stabilities of these 13 candidate reference genes under different conditions. Results showed that GAPDH and CYP expression remain stable throughout in the different abiotic stress treatments, CAC and PP2A expression were relatively stable under reproductive stages and α-TUB, PP2A and ETIF3E were found to be stably expressed in vegetative stages. Further, the expression levels of Diacylglycerol acyltransferase (DGAT1, a key enzyme in triacylglycerol synthesis was analyzed to confirm the validity of reference genes identified in the study. This is the first systematic study of selection of reference genes in S. hispanica, and will benefit future expression studies in this crop.

Normalisation genes for expression analyses in the brown alga model Ectocarpus siliculosus

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Rousvoal Sylvie

2008-08-01

Full Text Available Abstract Background Brown algae are plant multi-cellular organisms occupying most of the world coasts and are essential actors in the constitution of ecological niches at the shoreline. Ectocarpus siliculosus is an emerging model for brown algal research. Its genome has been sequenced, and several tools are being developed to perform analyses at different levels of cell organization, including transcriptomic expression analyses. Several topics, including physiological responses to osmotic stress and to exposure to contaminants and solvents are being studied in order to better understand the adaptive capacity of brown algae to pollution and environmental changes. A series of genes that can be used to normalise expression analyses is required for these studies. Results We monitored the expression of 13 genes under 21 different culture conditions. These included genes encoding proteins and factors involved in protein translation (ribosomal protein 26S, EF1alpha, IF2A, IF4E and protein degradation (ubiquitin, ubiquitin conjugating enzyme or folding (cyclophilin, and proteins involved in both the structure of the cytoskeleton (tubulin alpha, actin, actin-related proteins and its trafficking function (dynein, as well as a protein implicated in carbon metabolism (glucose 6-phosphate dehydrogenase. The stability of their expression level was assessed using the Ct range, and by applying both the geNorm and the Normfinder principles of calculation. Conclusion Comparisons of the data obtained with the three methods of calculation indicated that EF1alpha (EF1a was the best reference gene for normalisation. The normalisation factor should be calculated with at least two genes, alpha tubulin, ubiquitin-conjugating enzyme or actin-related proteins being good partners of EF1a. Our results exclude actin as a good normalisation gene, and, in this, are in agreement with previous studies in other organisms.

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.

Selection of Reliable Reference Genes for Gene Expression Studies of a Promising Oilseed Crop, Plukenetia volubilis, by Real-Time Quantitative PCR

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Longjian Niu

2015-06-01

Full Text Available Real-time quantitative PCR (RT-qPCR is a reliable and widely used method for gene expression analysis. The accuracy of the determination of a target gene expression level by RT-qPCR demands the use of appropriate reference genes to normalize the mRNA levels among different samples. However, suitable reference genes for RT-qPCR have not been identified in Sacha inchi (Plukenetia volubilis, a promising oilseed crop known for its polyunsaturated fatty acid (PUFA-rich seeds. In this study, using RT-qPCR, twelve candidate reference genes were examined in seedlings and adult plants, during flower and seed development and for the entire growth cycle of Sacha inchi. Four statistical algorithms (delta cycle threshold (ΔCt, BestKeeper, geNorm, and NormFinder were used to assess the expression stabilities of the candidate genes. The results showed that ubiquitin-conjugating enzyme (UCE, actin (ACT and phospholipase A22 (PLA were the most stable genes in Sacha inchi seedlings. For roots, stems, leaves, flowers, and seeds from adult plants, 30S ribosomal protein S13 (RPS13, cyclophilin (CYC and elongation factor-1alpha (EF1α were recommended as reference genes for RT-qPCR. During the development of reproductive organs, PLA, ACT and UCE were the optimal reference genes for flower development, whereas UCE, RPS13 and RNA polymerase II subunit (RPII were optimal for seed development. Considering the entire growth cycle of Sacha inchi, UCE, ACT and EF1α were sufficient for the purpose of normalization. Our results provide useful guidelines for the selection of reliable reference genes for the normalization of RT-qPCR data for seedlings and adult plants, for reproductive organs, and for the entire growth cycle of Sacha inchi.

Identification and Evaluation of Reliable Reference Genes in the Medicinal Fungus Shiraia bambusicola.

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Song, Liang; Li, Tong; Fan, Li; Shen, Xiao-Ye; Hou, Cheng-Lin

2016-04-01

The stability of reference genes plays a vital role in real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis, which is generally regarded as a convenient and sensitive tool for the analysis of gene expression. A well-known medicinal fungus, Shiraia bambusicola, has great potential in the pharmaceutical, agricultural and food industries, but its suitable reference genes have not yet been determined. In the present study, 11 candidate reference genes in S. bambusicola were first evaluated and validated comprehensively. To identify the suitable reference genes for qRT-PCR analysis, three software-based algorithms, geNorm, NormFinder and Best Keeper, were applied to rank the tested genes. RNA samples were collected from seven fermentation stages using different media (potato dextrose or Czapek medium) and under different light conditions (12-h light/12-h dark and all-dark). The three most appropriate reference genes, ubi, tfc and ags, were able to normalize the qRT-PCR results under the culturing conditions of 12-h light/12-h dark, whereas the other three genes, vac, gke and acyl, performed better in the culturing conditions of all-dark growth. Therefore, under different light conditions, at least two reference genes (ubi and vac) could be employed to assure the reliability of qRT-PCR results. For both the natural culture medium (the most appropriate genes of this group: ubi, tfc and ags) and the chemically defined synthetic medium (the most stable genes of this group: tfc, vac and ef), the tfc gene remained the best gene used for normalizing the gene expression found with qRT-PCR. It is anticipated that these results would improve the selection of suitable reference genes for qRT-PCR assays and lay the foundation for an accurate analysis of gene expression in S. bambusicola.

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 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.

A comprehensive approach to identify reliable reference gene candidates to investigate the link between alcoholism and endocrinology in Sprague-Dawley rats.

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Faten A Taki

Full Text Available Gender and hormonal differences are often correlated with alcohol dependence and related complications like addiction and breast cancer. Estrogen (E2 is an important sex hormone because it serves as a key protein involved in organism level signaling pathways. Alcoholism has been reported to affect estrogen receptor signaling; however, identifying the players involved in such multi-faceted syndrome is complex and requires an interdisciplinary approach. In many situations, preliminary investigations included a straight forward, yet informative biotechniques such as gene expression analyses using quantitative real time PCR (qRT-PCR. The validity of qRT-PCR-based conclusions is affected by the choice of reliable internal controls. With this in mind, we compiled a list of 15 commonly used housekeeping genes (HKGs as potential reference gene candidates in rat biological models. A comprehensive comparison among 5 statistical approaches (geNorm, dCt method, NormFinder, BestKeeper, and RefFinder was performed to identify the minimal number as well the most stable reference genes required for reliable normalization in experimental rat groups that comprised sham operated (SO, ovariectomized rats in the absence (OVX or presence of E2 (OVXE2. These rat groups were subdivided into subgroups that received alcohol in liquid diet or isocalroic control liquid diet for 12 weeks. Our results showed that U87, 5S rRNA, GAPDH, and U5a were the most reliable gene candidates for reference genes in heart and brain tissue. However, different gene stability ranking was specific for each tissue input combination. The present preliminary findings highlight the variability in reference gene rankings across different experimental conditions and analytic methods and constitute a fundamental step for gene expression assays.

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.

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.

Selection of Reliable Reference Genes for Gene Expression Studies of a Promising Oilseed Crop, Plukenetia volubilis, by Real-Time Quantitative PCR

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Niu, Longjian; Tao, Yan-Bin; Chen, Mao-Sheng; Fu, Qiantang; Li, Chaoqiong; Dong, Yuling; Wang, Xiulan; He, Huiying; Xu, Zeng-Fu

2015-01-01

Real-time quantitative PCR (RT-qPCR) is a reliable and widely used method for gene expression analysis. The accuracy of the determination of a target gene expression level by RT-qPCR demands the use of appropriate reference genes to normalize the mRNA levels among different samples. However, suitable reference genes for RT-qPCR have not been identified in Sacha inchi (Plukenetia volubilis), a promising oilseed crop known for its polyunsaturated fatty acid (PUFA)-rich seeds. In this study, using RT-qPCR, twelve candidate reference genes were examined in seedlings and adult plants, during flower and seed development and for the entire growth cycle of Sacha inchi. Four statistical algorithms (delta cycle threshold (ΔCt), BestKeeper, geNorm, and NormFinder) were used to assess the expression stabilities of the candidate genes. The results showed that ubiquitin-conjugating enzyme (UCE), actin (ACT) and phospholipase A22 (PLA) were the most stable genes in Sacha inchi seedlings. For roots, stems, leaves, flowers, and seeds from adult plants, 30S ribosomal protein S13 (RPS13), cyclophilin (CYC) and elongation factor-1alpha (EF1α) were recommended as reference genes for RT-qPCR. During the development of reproductive organs, PLA, ACT and UCE were the optimal reference genes for flower development, whereas UCE, RPS13 and RNA polymerase II subunit (RPII) were optimal for seed development. Considering the entire growth cycle of Sacha inchi, UCE, ACT and EF1α were sufficient for the purpose of normalization. Our results provide useful guidelines for the selection of reliable reference genes for the normalization of RT-qPCR data for seedlings and adult plants, for reproductive organs, and for the entire growth cycle of Sacha inchi. PMID:26047338

Selection of reference genes for expression analysis of Kumamoto and Portuguese oysters and their hybrid

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Yan, Lulu; Su, Jiaqi; Wang, Zhaoping; Yan, Xiwu; Yu, Ruihai

2017-12-01

Quantitative real-time polymerase chain reaction (qRT-PCR) is a rapid and reliable technique which has been widely used to quantifying gene transcripts (expression analysis). It is also employed for studying heterosis, hybridization breeding and hybrid tolerability of oysters, an ecologically and economically important taxonomic group. For these studies, selection of a suitable set of housekeeping genes as references is crucial for correct interpretation of qRT-PCR data. To identify suitable reference genes for oysters during low temperature and low salinity stresses, we analyzed twelve genes from the gill tissue of Crassostrea sikamea (SS), Crassostrea angulata (AA) and their hybrid (SA), which included three ribosomal genes, 28S ribosomal protein S5 ( RPS5), ribosomal protein L35 ( RPL35), and 60S ribosomal protein L29 ( RPL29); three structural genes, tubulin gamma ( TUBγ), annexin A6 and A7 ( AA6 and AA7); three metabolic pathway genes, ornithine decarboxylase ( OD), glyceraldehyde-3-phosphate dehydrogenase ( GAPDH) and glutathione S-transferase P1 ( GSP); two transcription factors, elongation factor 1 alpha and beta ( EF1α and EF1β); and one protein synthesis gene (ubiquitin ( UBQ). Primers specific for these genes were successfully developed for the three groups of oysters. Three different algorithms, geNorm, NormFinder and BestKeeper, were used to evaluate the expression stability of these candidate genes. BestKeeper program was found to be the most reliable. Based on our analysis, we found that the expression of RPL35 and EF1α was stable under low salinity stress, and the expression of OD, GAPDH and EF1α was stable under low temperature stress in hybrid (SA) oyster; the expression of RPS5 and GAPDH was stable under low salinity stress, and the expression of RPS5, UBQ, GAPDH was stable under low temperature stress in SS oyster; the expression of RPS5, GAPDH, EF1β and AA7 was stable under low salinity stress, and the expression of RPL35, EF1α, GAPDH

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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Xin Liu

2018-04-01

Full Text Available Background 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. Method 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. Result 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. Discussion

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

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.

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

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

Identification of miR-93 as a suitable miR for normalizing miRNA in plasma of tuberculosis patients.

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Barry, Simone E; Chan, Brian; Ellis, Magda; Yang, YuRong; Plit, Marshall L; Guan, Guangyu; Wang, Xiaolin; Britton, Warwick J; Saunders, Bernadette M

2015-07-01

Tuberculosis (TB) remains a major public health issue. New tests to aid diagnoses and monitor the response to therapy are urgently required. There is growing interest in the use of microRNA (miRNA) profiles as diagnostic, prognostic or predictive markers in a range of clinical and infectious diseases, including Mycobacterium tuberculosis infection, however, challenges exist to accurately normalise miRNA levels in cohorts. This study examined the appropriateness of 12 miRs and RNU6B to normalise circulating plasma miRNA levels in individuals with active TB from 2 different geographical and ethnic regions. Twelve miRs (let-7, miR-16, miR-22, miR-26, miR-93, miR-103, miR-191, miR-192, miR-221, miR-423, miR-425 and miR-451) and RNU6B were selected based on their reported production by lung cells, expression in blood and previous use as a reference miRNA. Expression levels were analysed in the plasma of newly diagnosed TB patients from Australia and China compared with individuals with latent TB infection and healthy volunteers. Analysis with both geNorm and NormFinder software identified miR-93 as the most suitable reference miR in both cohorts, either when analysed separately or collectively. Interestingly, there were large variations in the expression levels of some miRs, in particular miR-192 and let-7, between the two cohorts, independent of disease status. These data identify miR-93 is a suitable reference miR for normalizing miRNA levels in TB patients, and highlight how environmental, and possibly ethnic, factors influence miRNA expression levels, demonstrating the necessity of assessing the suitability of reference miRs within the study population. © 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Validation of suitable reference genes for expression normalization in Echinococcus spp. larval stages.

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Espínola, Sergio Martin; Ferreira, Henrique Bunselmeyer; Zaha, Arnaldo

2014-01-01

In recent years, a significant amount of sequence data (both genomic and transcriptomic) for Echinococcus spp. has been published, thereby facilitating the analysis of genes expressed during a specific stage or involved in parasite development. To perform a suitable gene expression quantification analysis, the use of validated reference genes is strongly recommended. Thus, the aim of this work was to identify suitable reference genes to allow reliable expression normalization for genes of interest in Echinococcus granulosus sensu stricto (s.s.) (G1) and Echinococcus ortleppi upon induction of the early pre-adult development. Untreated protoscoleces (PS) and pepsin-treated protoscoleces (PSP) from E. granulosus s.s. (G1) and E. ortleppi metacestode were used. The gene expression stability of eleven candidate reference genes (βTUB, NDUFV2, RPL13, TBP, CYP-1, RPII, EF-1α, βACT-1, GAPDH, ETIF4A-III and MAPK3) was assessed using geNorm, Normfinder, and RefFinder. Our qPCR data showed a good correlation with the recently published RNA-seq data. Regarding expression stability, EF-1α and TBP were the most stable genes for both species. Interestingly, βACT-1 (the most commonly used reference gene), and GAPDH and ETIF4A-III (previously identified as housekeeping genes) did not behave stably in our assay conditions. We propose the use of EF-1α as a reference gene for studies involving gene expression analysis in both PS and PSP experimental conditions for E. granulosus s.s. and E. ortleppi. To demonstrate its applicability, EF-1α was used as a normalizer gene in the relative quantification of transcripts from genes coding for antigen B subunits. The same EF-1α reference gene may be used in studies with other Echinococcus sensu lato species. This report validates suitable reference genes for species of class Cestoda, phylum Platyhelminthes, thus providing a foundation for further validation in other epidemiologically important cestode species, such as those from the

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.

Validation of Suitable Reference Genes for Expression Normalization in Echinococcus spp. Larval Stages

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Espínola, Sergio Martin; Ferreira, Henrique Bunselmeyer; Zaha, Arnaldo

2014-01-01

In recent years, a significant amount of sequence data (both genomic and transcriptomic) for Echinococcus spp. has been published, thereby facilitating the analysis of genes expressed during a specific stage or involved in parasite development. To perform a suitable gene expression quantification analysis, the use of validated reference genes is strongly recommended. Thus, the aim of this work was to identify suitable reference genes to allow reliable expression normalization for genes of interest in Echinococcus granulosus sensu stricto (s.s.) (G1) and Echinococcus ortleppi upon induction of the early pre-adult development. Untreated protoscoleces (PS) and pepsin-treated protoscoleces (PSP) from E. granulosus s.s. (G1) and E. ortleppi metacestode were used. The gene expression stability of eleven candidate reference genes (βTUB, NDUFV2, RPL13, TBP, CYP-1, RPII, EF-1α, βACT-1, GAPDH, ETIF4A-III and MAPK3) was assessed using geNorm, Normfinder, and RefFinder. Our qPCR data showed a good correlation with the recently published RNA-seq data. Regarding expression stability, EF-1α and TBP were the most stable genes for both species. Interestingly, βACT-1 (the most commonly used reference gene), and GAPDH and ETIF4A-III (previously identified as housekeeping genes) did not behave stably in our assay conditions. We propose the use of EF-1α as a reference gene for studies involving gene expression analysis in both PS and PSP experimental conditions for E. granulosus s.s. and E. ortleppi. To demonstrate its applicability, EF-1α was used as a normalizer gene in the relative quantification of transcripts from genes coding for antigen B subunits. The same EF-1α reference gene may be used in studies with other Echinococcus sensu lato species. This report validates suitable reference genes for species of class Cestoda, phylum Platyhelminthes, thus providing a foundation for further validation in other epidemiologically important cestode species, such as those from the

Validation of housekeeping genes for quantitative real-time PCR in in-vivo and in-vitro models of cerebral ischaemia

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Serena Joaquín

2009-06-01

Full Text Available Abstract Background Studies of gene expression in experimental cerebral ischaemia models can contribute to understanding the pathophysiology of brain ischaemia and to identifying prognostic markers and potential therapeutic targets. The normalization of relative qRT-PCR data using a suitable reference gene is a crucial prerequisite for obtaining reliable conclusions. No validated housekeeping genes have been reported for the relative quantification of the mRNA expression profile activated in in-vitro ischaemic conditions, whereas for the in-vivo model different reference genes have been used. The present study aims to determine the expression stability of ten housekeeping genes (Gapdh, β2m, Hprt, Ppia, Rpl13a, Oaz1, 18S rRNA, Gusb, Ywhaz and Sdha to establish their suitability as control genes for in-vitro and in-vivo cerebral ischaemia models. Results The expression stability of the candidate reference genes was evaluated using the 2-ΔC'T method and ANOVA followed by Dunnett's test. For the in-vitro model using primary cultures of rat astrocytes, all genes analysed except for Rpl13a and Sdha were found to have significantly different levels of mRNA expression. These different levels were also found in the case of the in-vivo model of pMCAO in rats except for Hprt, Sdha and Ywhaz mRNA, where the expression did not vary. Sdha and Ywhaz were identified by geNorm and NormFinder as the two most stable genes. Conclusion We have validated endogenous control genes for qRT-PCR analysis of gene expression in in-vitro and in-vivo cerebral ischaemia models. For normalization purposes, Rpl13a and Sdha are found to be the most suitable genes for the in-vitro model and Sdha and Ywhaz for the in-vivo model. Genes previously used as housekeeping genes for the in-vivo model in the literature were not validated as good control genes in the present study, showing the need for careful evaluation for each new experimental setup.

A Toolbox for Quantitative Gene Expression in Varroa destructor: RNA Degradation in Field Samples and Systematic Analysis of Reference Gene Stability.

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Ewan M Campbell

Full Text Available Varroa destructor is the major pest of Apis mellifera and contributes to the global honey bee health crisis threatening food security. Developing new control strategies to combat Varroa will require the application of molecular biology, including gene expression studies by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR. Both high quality RNA samples and suitable stable internal reference genes are required for accurate gene expression studies. In this study, ten candidate genes (succinate dehydrogenase (SDHA, NADH dehydrogenase (NADH, large ribsosmal subunit, TATA-binding protein, glyceraldehyde-3-phosphate dehydrogenase, 18S rRNA (18S, heat-shock protein 90 (HSP90, cyclophilin, α-tubulin, actin, were evaluated for their suitability as normalization genes using the geNorm, Normfinder, BestKeeper, and comparative ΔCq algorithims. Our study proposes the use of no more than two of the four most stable reference genes (NADH, 18S, SDHA and HSP90 in Varroa gene expression studies. These four genes remain stable in phoretic and reproductive stage Varroa and are unaffected by Deformed wing virus load. When used for determining changes in vitellogenin gene expression, the signal-to-noise ratio (SNR for the relatively unstable genes actin and α-tubulin was much lower than for the stable gene combinations (NADH + HSP90 +18S; NADH + HSP90; or NADH. Using both electropherograms and RT-qPCR for short and long amplicons as quality controls, we demonstrate that high quality RNA can be recovered from Varroa up to 10 days later stored at ambient temperature if collected into RNAlater and provided the body is pierced. This protocol allows the exchange of Varroa samples between international collaborators and field sample collectors without requiring frozen collection or shipping. Our results make important contributions to gene expression studies in Varroa by proposing a validated sampling protocol to obtain high quality Varroa

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.

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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

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

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

Validation of reference genes from Eucalyptus spp. under different stress conditions

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Moura Jullyana Cristina Magalhães Silva

2012-11-01

Full Text Available Abstract Background The genus Eucalyptus consists of approximately 600 species and subspecies and has a physiological plasticity that allows some species to propagate in different regions of the world. Eucalyptus is a major source of cellulose for paper manufacturing, and its cultivation is limited by weather conditions, particularly water stress and low temperatures. Gene expression studies using quantitative reverse transcription polymerase chain reaction (qPCR require reference genes, which must have stable expression to facilitate the comparison of the results from analyses using different species, tissues, and treatments. Such studies have been limited in eucalyptus. Results Eucalyptus globulus Labill, Eucalyptus urograndis (hybrid from Eucalyptus urophylla S.T. Blake X Eucalyptus grandis Hill ex-Maiden and E. uroglobulus (hybrid from E. urograndis X E. globulus were subjected to different treatments, including water deficiency and stress recovery, low temperatures, presence or absence of light, and their respective controls. Except for treatment with light, which examined the seedling hypocotyl or apical portion of the stem, the expression analyses were conducted in the apical and basal parts of the stem. To select the best pair of genes, the bioinformatics tools GeNorm and NormFinder were compared. Comprehensive analyses that did not differentiate between species, treatments, or tissue types, showed that IDH (isocitrate dehydrogenase, SAND (SAND protein, ACT (actin, and A-Tub (α-tubulin genes were the most stable. IDH was the most stable gene in all of the treatments. Conclusion Comparing these results with those of other studies on eucalyptus, we concluded that five genes are stable in different species and experimental conditions: IDH, SAND, ACT, A-Tub, and UBQ (ubiquitin. It is usually recommended a minimum of two reference genes is expression analysis; therefore, we propose that IDH and two others genes among the five identified

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

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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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

Reference gene validation for gene expression normalization in canine osteosarcoma : a geNorm algorithm approach

NARCIS (Netherlands)

Selvarajah, G.T.; Bonestroo, F.A.S.; Timmermans Sprang, E.P.M.; Kirpensteijn, J.|info:eu-repo/dai/nl/189846992; Mol, J.A.|info:eu-repo/dai/nl/070918775

2017-01-01

Background Quantitative PCR (qPCR) is a common method for quantifying mRNA expression. Given the heterogeneity present in tumor tissues, it is crucial to normalize target mRNA expression data using appropriate reference genes that are stably expressed under a variety of pathological and experimental

Identification of housekeeping genes as references for quantitative ...

Indian Academy of Sciences (India)

XIAOHUA XIA

2017-11-27

Nov 27, 2017 ... required for the maintenance of basic cellular function. Generally, housekeeping genes are ... out to assess different housekeeping genes in Misgur- ... Data were eval- uated by three methods, geNorm (Vandesompele et al.

Reference genes for real-time PCR quantification of messenger RNAs and microRNAs in mouse model of obesity.

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Matoušková, Petra; Bártíková, Hana; Boušová, Iva; Hanušová, Veronika; Szotáková, Barbora; Skálová, Lenka

2014-01-01

Obesity and metabolic syndrome is increasing health problem worldwide. Among other ways, nutritional intervention using phytochemicals is important method for treatment and prevention of this disease. Recent studies have shown that certain phytochemicals could alter the expression of specific genes and microRNAs (miRNAs) that play a fundamental role in the pathogenesis of obesity. For study of the obesity and its treatment, monosodium glutamate (MSG)-injected mice with developed central obesity, insulin resistance and liver lipid accumulation are frequently used animal models. To understand the mechanism of phytochemicals action in obese animals, the study of selected genes expression together with miRNA quantification is extremely important. For this purpose, real-time quantitative PCR is a sensitive and reproducible method, but it depends on proper normalization entirely. The aim of present study was to identify the appropriate reference genes for mRNA and miRNA quantification in MSG mice treated with green tea catechins, potential anti-obesity phytochemicals. Two sets of reference genes were tested: first set contained seven commonly used genes for normalization of messenger RNA, the second set of candidate reference genes included ten small RNAs for normalization of miRNA. The expression stability of these reference genes were tested upon treatment of mice with catechins using geNorm, NormFinder and BestKeeper algorithms. Selected normalizers for mRNA quantification were tested and validated on expression of quinone oxidoreductase, biotransformation enzyme known to be modified by catechins. The effect of selected normalizers for miRNA quantification was tested on two obesity- and diabetes- related miRNAs, miR-221 and miR-29b, respectively. Finally, the combinations of B2M/18S/HPRT1 and miR-16/sno234 were validated as optimal reference genes for mRNA and miRNA quantification in liver and 18S/RPlP0/HPRT1 and sno234/miR-186 in small intestine of MSG mice. These

Validation of putative reference genes for gene expression studies in human hepatocellular carcinoma using real-time quantitative RT-PCR

International Nuclear Information System (INIS)

Cicinnati, Vito R; Shen, Qingli; Sotiropoulos, Georgios C; Radtke, Arnold; Gerken, Guido; Beckebaum, Susanne

2008-01-01

Reference genes, which are often referred to as housekeeping genes are frequently used to normalize mRNA levels between different samples in quantitative reverse transcription polymerase chain reaction (qRT-PCR). The selection of reference genes is critical for gene expression studies because the expression of these genes may vary among tissues or cells and may change under certain circumstances. Here, a systematic evaluation of six putative reference genes for gene expression studies in human hepatocellular carcinoma (HCC) is presented. Six genes, beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethyl-bilane synthase (HMBS), hypoxanthine phosphoribosyl-transferase 1 (HPRT1), succinate dehydrogenase complex, subunit A (SDHA) and ubiquitin C (UBC), with distinct functional characteristics and expression patterns were evaluated by qRT-PCR. Inhibitory substances in RNA samples were quantitatively assessed and controlled using an external RNA control. The stability of selected reference genes was analyzed using both geNorm and NormFinder software. HMBS and GAPDH were identified as the optimal reference genes for normalizing gene expression data between paired tumoral and adjacent non-tumoral tissues derived from patients with HCC. HMBS, GAPDH and UBC were identified to be suitable for the normalization of gene expression data among tumor tissues; whereas the combination of HMBS, B2M, SDHA and GAPDH was suitable for normalizing gene expression data among five liver cancer cell lines, namely Hep3B, HepG2, HuH7, SK-HEP-1 and SNU-182. The determined gene stability was increased after exclusion of RNA samples containing relatively higher inhibitory substances. Of six genes studied, HMBS was found to be the single best reference gene for gene expression studies in HCC. The appropriate choice of combination of more than one reference gene to improve qRT-PCR accuracy depends on the kind of liver tissues or cells under investigation

Identification of Phosphoglycerate Kinase 1 (PGK1 as a reference gene for quantitative gene expression measurements in human blood RNA

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Unger Elizabeth R

2011-09-01

Full Text Available Abstract Background Blood is a convenient sample and increasingly used for quantitative gene expression measurements with a variety of diseases including chronic fatigue syndrome (CFS. Quantitative gene expression measurements require normalization of target genes to reference genes that are stable and independent from variables being tested in the experiment. Because there are no genes that are useful for all situations, reference gene selection is an essential step to any quantitative reverse transcription-PCR protocol. Many publications have described appropriate genes for a wide variety of tissues and experimental conditions, however, reference genes that may be suitable for the analysis of CFS, or human blood RNA derived from whole blood as well as isolated peripheral blood mononuclear cells (PBMCs, have not been described. Findings Literature review and analyses of our unpublished microarray data were used to narrow down the pool of candidate reference genes to six. We assayed whole blood RNA from Tempus tubes and cell preparation tube (CPT-collected PBMC RNA from 46 subjects, and used the geNorm and NormFinder algorithms to select the most stable reference genes. Phosphoglycerate kinase 1 (PGK1 was one of the optimal normalization genes for both whole blood and PBMC RNA, however, additional genes differed for the two sample types; Ribosomal protein large, P0 (RPLP0 for PBMC RNA and Peptidylprolyl isomerase B (PPIB for whole blood RNA. We also show that the use of a single reference gene is sufficient for normalization when the most stable candidates are used. Conclusions We have identified PGK1 as a stable reference gene for use with whole blood RNA and RNA derived from PBMC. When stable genes are selected it is possible to use a single gene for normalization rather than two or three. Optimal normalization will improve the ability of results from PBMC RNA to be compared with those from whole blood RNA and potentially allows comparison 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

Validation of putative reference genes for gene expression studies in human hepatocellular carcinoma using real-time quantitative RT-PCR

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Radtke Arnold

2008-11-01

Full Text Available Abstract Background Reference genes, which are often referred to as housekeeping genes are frequently used to normalize mRNA levels between different samples in quantitative reverse transcription polymerase chain reaction (qRT-PCR. The selection of reference genes is critical for gene expression studies because the expression of these genes may vary among tissues or cells and may change under certain circumstances. Here, a systematic evaluation of six putative reference genes for gene expression studies in human hepatocellular carcinoma (HCC is presented. Methods Six genes, beta-2-microglobulin (B2M, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, hydroxymethyl-bilane synthase (HMBS, hypoxanthine phosphoribosyl-transferase 1 (HPRT1, succinate dehydrogenase complex, subunit A (SDHA and ubiquitin C (UBC, with distinct functional characteristics and expression patterns were evaluated by qRT-PCR. Inhibitory substances in RNA samples were quantitatively assessed and controlled using an external RNA control. The stability of selected reference genes was analyzed using both geNorm and NormFinder software. Results HMBS and GAPDH were identified as the optimal reference genes for normalizing gene expression data between paired tumoral and adjacent non-tumoral tissues derived from patients with HCC. HMBS, GAPDH and UBC were identified to be suitable for the normalization of gene expression data among tumor tissues; whereas the combination of HMBS, B2M, SDHA and GAPDH was suitable for normalizing gene expression data among five liver cancer cell lines, namely Hep3B, HepG2, HuH7, SK-HEP-1 and SNU-182. The determined gene stability was increased after exclusion of RNA samples containing relatively higher inhibitory substances. Conclusion Of six genes studied, HMBS was found to be the single best reference gene for gene expression studies in HCC. The appropriate choice of combination of more than one reference gene to improve qRT-PCR accuracy depends on the

Validation of putative reference genes for gene expression studies in human hepatocellular carcinoma using real-time quantitative RT-PCR

Science.gov (United States)

Cicinnati, Vito R; Shen, Qingli; Sotiropoulos, Georgios C; Radtke, Arnold; Gerken, Guido; Beckebaum, Susanne

2008-01-01

Background Reference genes, which are often referred to as housekeeping genes are frequently used to normalize mRNA levels between different samples in quantitative reverse transcription polymerase chain reaction (qRT-PCR). The selection of reference genes is critical for gene expression studies because the expression of these genes may vary among tissues or cells and may change under certain circumstances. Here, a systematic evaluation of six putative reference genes for gene expression studies in human hepatocellular carcinoma (HCC) is presented. Methods Six genes, beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethyl-bilane synthase (HMBS), hypoxanthine phosphoribosyl-transferase 1 (HPRT1), succinate dehydrogenase complex, subunit A (SDHA) and ubiquitin C (UBC), with distinct functional characteristics and expression patterns were evaluated by qRT-PCR. Inhibitory substances in RNA samples were quantitatively assessed and controlled using an external RNA control. The stability of selected reference genes was analyzed using both geNorm and NormFinder software. Results HMBS and GAPDH were identified as the optimal reference genes for normalizing gene expression data between paired tumoral and adjacent non-tumoral tissues derived from patients with HCC. HMBS, GAPDH and UBC were identified to be suitable for the normalization of gene expression data among tumor tissues; whereas the combination of HMBS, B2M, SDHA and GAPDH was suitable for normalizing gene expression data among five liver cancer cell lines, namely Hep3B, HepG2, HuH7, SK-HEP-1 and SNU-182. The determined gene stability was increased after exclusion of RNA samples containing relatively higher inhibitory substances. Conclusion Of six genes studied, HMBS was found to be the single best reference gene for gene expression studies in HCC. The appropriate choice of combination of more than one reference gene to improve qRT-PCR accuracy depends on the kind of liver tissues

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

Directory of Open Access Journals (Sweden)

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

Reference genes for normalization: A study of rat brain tissue

DEFF Research Database (Denmark)

Bonefeld, Birgit; Elfving, Betina; Wegener, Gregers

2008-01-01

are warranted. With the overall aim to inspect the gene expression of three target genes, NMDAR1, SORT, and CREB, in rat hippocampus, we tested a panel of eight HKGs, 18s rRNA, ActB, CycA, Gapd, Hmbs, Hprt1, Rpl13A, and Ywhaz in order to select the most stably expressed gene, using the NormFinder and ge...

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

Directory of Open Access Journals (Sweden)

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

Expression stability and selection of optimal reference genes for gene expression normalization in early life stage rainbow trout exposed to cadmium and copper.

Science.gov (United States)

Shekh, Kamran; Tang, Song; Niyogi, Som; Hecker, Markus

2017-09-01

Gene expression analysis represents a powerful approach to characterize the specific mechanisms by which contaminants interact with organisms. One of the key considerations when conducting gene expression analyses using quantitative real-time reverse transcription-polymerase chain reaction (qPCR) is the selection of appropriate reference genes, which is often overlooked. Specifically, to reach meaningful conclusions when using relative quantification approaches, expression levels of reference genes must be highly stable and cannot vary as a function of experimental conditions. However, to date, information on the stability of commonly used reference genes across developmental stages, tissues and after exposure to contaminants such as metals is lacking for many vertebrate species including teleost fish. Therefore, in this study, we assessed the stability of expression of 8 reference gene candidates in the gills and skin of three different early life-stages of rainbow trout after acute exposure (24h) to two metals, cadmium (Cd) and copper (Cu) using qPCR. Candidate housekeeping genes were: beta actin (b-actin), DNA directed RNA polymerase II subunit I (DRP2), elongation factor-1 alpha (EF1a), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), glucose-6-phosphate dehydrogenase (G6PD), hypoxanthine phosphoribosyltransferase (HPRT), ribosomal protein L8 (RPL8), and 18S ribosomal RNA (18S). Four algorithms, geNorm, NormFinder, BestKeeper, and the comparative ΔCt method were employed to systematically evaluate the expression stability of these candidate genes under control and exposed conditions as well as across three different life-stages. Finally, stability of genes was ranked by taking geometric means of the ranks established by the different methods. Stability of reference genes was ranked in the following order (from lower to higher stability): HPRT

Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR

DEFF Research Database (Denmark)

Hillig, Ann-Britt Nygaard; Jørgensen, Claus Bøttcher; Cirera, Susanna

2007-01-01

-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethylbilane synthase (HMBS), hypoxanthine phosphoribosyltransferase I (HPRT I), ribosomal protein L4 (RPL4), succinate dehydrogenase complex subunit A (SDHA), TATA box binding protein (TPB) and tyrosine 3-monooxygenase/tryptophan 5......-monooxygenase activation protein zeta polypeptide (YWHAZ). The stability of these reference genes in different pig tissues was investigated using the geNorm application. The range of expression stability in the genes analysed was (from the most stable to the least stable): ACTB/RPL4, TBP, HPRT, HMBS, YWHAZ...

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

Directory of Open Access Journals (Sweden)

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.

Science.gov (United States)

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

Behavioral program synthesis with genetic programming

CERN Document Server

Krawiec, Krzysztof

2016-01-01

Genetic programming (GP) is a popular heuristic methodology of program synthesis with origins in evolutionary computation. In this generate-and-test approach, candidate programs are iteratively produced and evaluated. The latter involves running programs on tests, where they exhibit complex behaviors reflected in changes of variables, registers, or memory. That behavior not only ultimately determines program output, but may also reveal its `hidden qualities' and important characteristics of the considered synthesis problem. However, the conventional GP is oblivious to most of that information and usually cares only about the number of tests passed by a program. This `evaluation bottleneck' leaves search algorithm underinformed about the actual and potential qualities of candidate programs. This book proposes behavioral program synthesis, a conceptual framework that opens GP to detailed information on program behavior in order to make program synthesis more efficient. Several existing and novel mechanisms subs...

Selection of suitable reference genes for normalization of genes of interest in canine soft tissue sarcomas using quantitative real-time polymerase chain reaction

DEFF Research Database (Denmark)

Zornhagen, K. W.; Kristensen, A. T.; Hansen, Anders Elias

2015-01-01

Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) is a sensitive technique for quantifying gene expression. Stably expressed reference genes are necessary for normalization of RT-qPCR data. Only a few articles have been published on reference genes in canine tumours....... The objective of this study was to demonstrate how to identify suitable reference genes for normalization of genes of interest in canine soft tissue sarcomas using RT-qPCR. Primer pairs for 17 potential reference genes were designed and tested in archival tumour biopsies from six dogs. The geNorm algorithm...

Functional Programming

OpenAIRE

Chitil, Olaf

2009-01-01

Functional programming is a programming paradigm like object-oriented programming and logic programming. Functional programming comprises both a specific programming style and a class of programming languages that encourage and support this programming style. Functional programming enables the programmer to describe an algorithm on a high-level, in terms of the problem domain, without having to deal with machine-related details. A program is constructed from functions that only map inputs to ...

Programming language for computations in the Interkosmos program

Science.gov (United States)

Schmidt, K.

1975-01-01

The programming system for Intercosmos data processing, based on the structural programming theory, which considers a program as an ordered set of standardized elementary parts, from which the user programs are automatically generated, is described. The programs are comprised of several modules, which are briefly summarized. The general structure of the programming system is presented in a block diagram. A programming control language developed to formulate the problem quickly and completely is presented along with basic symbols which are characteristic of the Intercosmos programming system.

Maine Migrant Program: 1997-1998 Program Evaluation.

Science.gov (United States)

Bazinet, Suzanne C., Ed.

The Maine Department of Education contracts with local educational agencies to administer the Maine Migrant Education Program. The program's overall mission is to provide the support necessary for migrant children to achieve Maine's academic standards. In 1997-98, 73 local migrant programs served 9,838 students, and 63 summer programs served 1,769…

Program Leadership from a Nordic Perspective - Program Leaders' Power to Influence Their Program

DEFF Research Database (Denmark)

Högfeldt, Anna-Karin; Strömberg, Emma; Jerbrant, Anna

2013-01-01

research demonstrated that program leaders have quite different positions, strategies and methods when it comes to monitoring and developing their programs. In this paper, a deeper investigation is carried out of the (im-) possibilities to make real influence on the study courses that constitutes...... the respective Engineering study programs. Eight program leaders from the five N5T universities have been interviewed, and the analysis of these studies, has culminated in a model for the analysis of program leadership for Engineering education development....

Multi-Year Program Plan - Building Regulatory Programs

Energy Technology Data Exchange (ETDEWEB)

none,

2010-10-01

This document presents DOE’s multi-year plan for the three components of the Buildings Regulatory Program: Appliance and Equipment Efficiency Standards, ENERGY STAR, and the Building Energy Codes Program. This document summarizes the history of these programs, the mission and goals of the programs, pertinent statutory requirements, and DOE’s 5-year plan for moving forward.

Program summary for the Civilian Reactor Development Program

International Nuclear Information System (INIS)

1982-07-01

This Civilian Reactor Development Program document has the prime purpose of summarizing the technical programs supported by the FY 1983 budget request. This section provides a statement of the overall program objectives and a general program overview. Section II presents the technical programs in a format intended to show logical technical interrelationships, and does not necessarily follow the structure of the formal budget presentation. Section III presents the technical organization and management structure of the program

Geothermal Technologies Program Overview - Peer Review Program

Energy Technology Data Exchange (ETDEWEB)

Milliken, JoAnn [Office of Energy Efficiency and Renewable Energy (EERE), Washington, DC (United States)

2011-06-06

This Geothermal Technologies Program presentation was delivered on June 6, 2011 at a Program Peer Review meeting. It contains annual budget, Recovery Act, funding opportunities, upcoming program activities, and more.

A Program Transformation for Backwards Analysis of Logic Programs

DEFF Research Database (Denmark)

Gallagher, John Patrick

2003-01-01

The input to backwards analysis is a program together with properties that are required to hold at given program points. The purpose of the analysis is to derive initial goals or pre-conditions that guarantee that, when the program is executed, the given properties hold. The solution for logic...... programs presented here is based on a transformation of the input program, which makes explicit the dependencies of the given program points on the initial goals. The transformation is derived from the resultants semantics of logic programs. The transformed program is then analysed using a standard...

Object-Oriented Programming in the Beta Programming Language

DEFF Research Database (Denmark)

Madsen, Ole Lehrmann; Møller-Pedersen, Birger; Nygaard, Kristen

This is a book on object-oriented programming and the BETA programming language. Object-oriented programming originated with the Simula languages developed at the Norwegian Computing Center, Oslo, in the 1960s. The first Simula language, Simula I, was intended for writing simulation programs....... Simula I was later used as a basis for defining a general purpose programming language, Simula 67. In addition to being a programming language, Simula1 was also designed as a language for describing and communicating about systems in general. Simula has been used by a relatively small community for many...... years, although it has had a major impact on research in computer science. The real breakthrough for object-oriented programming came with the development of Smalltalk. Since then, a large number of programming languages based on Simula concepts have appeared. C++ is the language that has had...

Repository-Based Software Engineering Program: Working Program Management Plan

Science.gov (United States)

1993-01-01

Repository-Based Software Engineering Program (RBSE) is a National Aeronautics and Space Administration (NASA) sponsored program dedicated to introducing and supporting common, effective approaches to software engineering practices. The process of conceiving, designing, building, and maintaining software systems by using existing software assets that are stored in a specialized operational reuse library or repository, accessible to system designers, is the foundation of the program. In addition to operating a software repository, RBSE promotes (1) software engineering technology transfer, (2) academic and instructional support of reuse programs, (3) the use of common software engineering standards and practices, (4) software reuse technology research, and (5) interoperability between reuse libraries. This Program Management Plan (PMP) is intended to communicate program goals and objectives, describe major work areas, and define a management report and control process. This process will assist the Program Manager, University of Houston at Clear Lake (UHCL) in tracking work progress and describing major program activities to NASA management. The goal of this PMP is to make managing the RBSE program a relatively easy process that improves the work of all team members. The PMP describes work areas addressed and work efforts being accomplished by the program; however, it is not intended as a complete description of the program. Its focus is on providing management tools and management processes for monitoring, evaluating, and administering the program; and it includes schedules for charting milestones and deliveries of program products. The PMP was developed by soliciting and obtaining guidance from appropriate program participants, analyzing program management guidance, and reviewing related program management documents.

Program auto

International Nuclear Information System (INIS)

Rawool-Sullivan, M.W.; Plagnol, E.

1990-01-01

The program AUTO was developed to be used in the analysis of dE vs E type spectra. This program is written in FORTRAN and calculates dE vs E lines in MeV. The provision is also made in the program to convert these lines from MeV to ADC channel numbers to facilitate the comparison with the raw data from the experiments. Currently the output of this program can be plotted with the display program, called VISU, but it can also be used independent of the program VISU, with little or no modification in the actual fortran code. The program AUTO has many useful applications. In this article the program AUTO is described along with its applications

A Playful Programming Products Vs. Programming Concepts Matrix

DEFF Research Database (Denmark)

Allsopp, Benjamin Brink

2017-01-01

to computer program: playful programming. This research also describes a project to bring together different stakeholders (developers, educators, parents and researchers) with a common vocabulary for describing, developing, teaching with and comparing these playful programming products and develops a model......A number of Danish primary schools are involved in pilot studies where 1st to 9th grade students work with Scratch and Lego MindStorms in STEM subjects. These games may become part of the curriculum at these schools. Recent research identifies a category of games and toys that support learning...... to provide educators and researchers involved in pilot studies with an overview of which programming concepts various playful programming products exercise (a playful programming products vs. programming concepts matrix). We also add additional concept specializations and expand on the descriptions...

Programming by Numbers -- A Programming Method for Complete Novices

NARCIS (Netherlands)

Glaser, Hugh; Hartel, Pieter H.

2000-01-01

Students often have difficulty with the minutiae of program construction. We introduce the idea of `Programming by Numbers', which breaks some of the programming process down into smaller steps, giving such students a way into the process of Programming in the Small. Programming by Numbers does not

Clean Coal Technology Demonstration Program: Program Update 1998

Energy Technology Data Exchange (ETDEWEB)

Assistant Secretary for Fossil Energy

1999-03-01

Annual report on the Clean Coal Technology Demonstration Program (CCT Program). The report address the role of the CCT Program, implementation, funding and costs, accomplishments, project descriptions, legislative history, program history, environmental aspects, and project contacts. The project descriptions describe the technology and provides a brief summary of the demonstration results.

Program specialization

CERN Document Server

Marlet, Renaud

2013-01-01

This book presents the principles and techniques of program specialization - a general method to make programs faster (and possibly smaller) when some inputs can be known in advance. As an illustration, it describes the architecture of Tempo, an offline program specializer for C that can also specialize code at runtime, and provides figures for concrete applications in various domains. Technical details address issues related to program analysis precision, value reification, incomplete program specialization, strategies to exploit specialized program, incremental specialization, and data speci

UNDERSTANDING THE RELATIONSHIPS OF PROGRAM SATISFACTION, PROGRAM LOYALTY AND STORE LOYALTY AMONG CARDHOLDERS OF LOYALTY PROGRAMS

Directory of Open Access Journals (Sweden)

Nor Asiah Omar

2011-01-01

Full Text Available Loyalty programs have increasingly attracted interest in both academic marketing research and practice. One major factor that has been increasingly discussed is loyalty. In this study we examine the influence of cardholders' satisfaction on loyalty (program loyalty and store loyalty in a retail context, namely, in department stores and superstores. Data were collected from 400 cardholders of a retail loyalty program in Klang Valley, Malaysia via the drop-off-and-collect technique. Structural modelling techniques were applied to analyze the data. The results indicated that program satisfaction is not related to store loyalty (share-of-wallet, share-of-visit and store preference. However, loyalty to the program (program loyalty plays a crucial intervening role in the relationship between program satisfaction and store loyalty. The study underscores the principal importance of program loyalty in the retail loyalty program.

Collectively loading programs in a multiple program multiple data environment

Science.gov (United States)

Aho, Michael E.; Attinella, John E.; Gooding, Thomas M.; Gooding, Thomas M.; Miller, Samuel J.

2016-11-08

Techniques are disclosed for loading programs efficiently in a parallel computing system. In one embodiment, nodes of the parallel computing system receive a load description file which indicates, for each program of a multiple program multiple data (MPMD) job, nodes which are to load the program. The nodes determine, using collective operations, a total number of programs to load and a number of programs to load in parallel. The nodes further generate a class route for each program to be loaded in parallel, where the class route generated for a particular program includes only those nodes on which the program needs to be loaded. For each class route, a node is selected using a collective operation to be a load leader which accesses a file system to load the program associated with a class route and broadcasts the program via the class route to other nodes which require the program.

Clean Coal Technology Demonstration Program: Program Update 2001

Energy Technology Data Exchange (ETDEWEB)

Assistant Secretary for Fossil Energy

2002-07-30

Annual report on the Clean Coal Technology Demonstration Program (CCT Program). The report address the role of the CCT Program, implementation, funding and costs, accomplishments, project descriptions, legislative history, program history, environmental aspects, and project contacts. The project descriptions describe the technology and provides a brief summary of the demonstration results. Also includes Power Plant Improvement Initiative Projects.

An Analysis of Programming Beginners' Source Programs

Science.gov (United States)

Matsuyama, Chieko; Nakashima, Toyoshiro; Ishii, Naohiro

The production of animations was made the subject of a university programming course in order to make students understand the process of program creation, and so that students could tackle programming with interest. In this paper, the formats and composition of the programs which students produced were investigated. As a result, it was found that there were a lot of problems related to such matters as how to use indent, how to apply comments and functions etc. for the format and the composition of the source codes.

Learners Programming Language a Helping System for Introductory Programming Courses

Directory of Open Access Journals (Sweden)

MUHAMMAD SHUMAIL NAVEED

2016-07-01

Full Text Available Programming is the core of computer science and due to this momentousness a special care is taken in designing the curriculum of programming courses. A substantial work has been conducted on the definition of programming courses, yet the introductory programming courses are still facing high attrition, low retention and lack of motivation. This paper introduced a tiny pre-programming language called LPL (Learners Programming Language as a ZPL (Zeroth Programming Language to illuminate novice students about elementary concepts of introductory programming before introducing the first imperative programming course. The overall objective and design philosophy of LPL is based on a hypothesis that the soft introduction of a simple and paradigm specific textual programming can increase the motivation level of novice students and reduce the congenital complexities and hardness of the first programming course and eventually improve the retention rate and may be fruitful in reducing the dropout/failure level. LPL also generates the equivalent high level programs from user source program and eventually very fruitful in understanding the syntax of introductory programming languages. To overcome the inherent complexities of unusual and rigid syntax of introductory programming languages, the LPL provide elementary programming concepts in the form of algorithmic and plain natural language based computational statements. The initial results obtained after the introduction of LPL are very encouraging in motivating novice students and improving the retention rate.

Analyzing Array Manipulating Programs by Program Transformation

Science.gov (United States)

Cornish, J. Robert M.; Gange, Graeme; Navas, Jorge A.; Schachte, Peter; Sondergaard, Harald; Stuckey, Peter J.

2014-01-01

We explore a transformational approach to the problem of verifying simple array-manipulating programs. Traditionally, verification of such programs requires intricate analysis machinery to reason with universally quantified statements about symbolic array segments, such as "every data item stored in the segment A[i] to A[j] is equal to the corresponding item stored in the segment B[i] to B[j]." We define a simple abstract machine which allows for set-valued variables and we show how to translate programs with array operations to array-free code for this machine. For the purpose of program analysis, the translated program remains faithful to the semantics of array manipulation. Based on our implementation in LLVM, we evaluate the approach with respect to its ability to extract useful invariants and the cost in terms of code size.

Subseabed-disposal program: systems-analysis program plan

International Nuclear Information System (INIS)

Klett, R.D.

1981-03-01

This report contains an overview of the Subseabed Nuclear Waste Disposal Program systems analysis program plan, and includes sensitivity, safety, optimization, and cost/benefit analyses. Details of the primary barrier sensitivity analysis and the data acquisition and modeling cost/benefit studies are given, as well as the schedule through the technical, environmental, and engineering feasibility phases of the program

Federal Wind Energy Program. Program summary. [USA

Energy Technology Data Exchange (ETDEWEB)

None

1978-01-01

The objective of the Federal Wind Energy Program is to accelerate the development of reliable and economically viable wind energy systems and enable the earliest possible commercialization of wind power. To achieve this objective for small and large wind systems requires advancing the technology, developing a sound industrial technology base, and addressing the non-technological issues which could deter the use of wind energy. This summary report outlines the projects being supported by the program through FY 1977 toward the achievement of these goals. It also outlines the program's general organization and specific program elements.

Automatic Construction of Java Programs from Functional Program Specifications

OpenAIRE

Md. Humayun Kabir

2015-01-01

This paper presents a novel approach to construct Java programs automatically from the input functional program specifications on natural numbers from the constructive proofs of the input specifications using an inductive theorem prover called Poiti'n. The construction of a Java program from the input functional program specification involves two phases. The theorem prover is used to construct a higher order functional (HOF) program from the input specification expressed as an existential the...

Environmental radioactive intercomparison program and radioactive standards program

Energy Technology Data Exchange (ETDEWEB)

Dilbeck, G. [Environmental Monitoring Systems Laboratory, Las Vegas, NV (United States)

1993-12-31

The Environmental Radioactivity Intercomparison Program described herein provides quality assurance support for laboratories involved in analyzing public drinking water under the Safe Drinking Water Act (SDWA) Regulations, and to the environmental radiation monitoring activities of various agencies. More than 300 federal and state nuclear facilities and private laboratories participate in some phase of the program. This presentation describes the Intercomparison Program studies and matrices involved, summarizes the precision and accuracy requirements of various radioactive analytes, and describes the traceability determinations involved with radioactive calibration standards distributed to the participants. A summary of program participants, sample and report distributions, and additional responsibilities of this program are discussed.

Veterinary Technician Program Director Leadership Style and Program Success

Science.gov (United States)

Renda-Francis, Lori A.

2012-01-01

Program directors of American Veterinary Medical Association (AVMA) accredited veterinary technician programs may have little or no training in leadership. The need for program directors of AVMA-accredited veterinary technician programs to understand how leadership traits may have an impact on student success is often overlooked. The purpose of…

Programming

International Nuclear Information System (INIS)

Jackson, M.A.

1982-01-01

The programmer's task is often taken to be the construction of algorithms, expressed in hierarchical structures of procedures: this view underlies the majority of traditional programming languages, such as Fortran. A different view is appropriate to a wide class of problem, perhaps including some problems in High Energy Physics. The programmer's task is regarded as having three main stages: first, an explicit model is constructed of the reality with which the program is concerned; second, this model is elaborated to produce the required program outputs; third, the resulting program is transformed to run efficiently in the execution environment. The first two stages deal in network structures of sequential processes; only the third is concerned with procedure hierarchies. (orig.)

Knowledge, programming, and programming cultures: LISP, C, and Ada

Science.gov (United States)

Rochowiak, Daniel

1990-01-01

The results of research 'Ada as an implementation language for knowledge based systems' are presented. The purpose of the research was to compare Ada to other programming languages. The report focuses on the programming languages Ada, C, and Lisp, the programming cultures that surround them, and the programming paradigms they support.

System programming languages

OpenAIRE

Šmit, Matej

2016-01-01

Most operating systems are written in the C programming language. Similar is with system software, for example, device drivers, compilers, debuggers, disk checkers, etc. Recently some new programming languages emerged, which are supposed to be suitable for system programming. In this thesis we present programming languages D, Go, Nim and Rust. We defined the criteria which are important for deciding whether programming language is suitable for system programming. We examine programming langua...

Identification of potential internal control genes for real-time PCR analysis during stress response in Pyropia haitanensis

Science.gov (United States)

Wang, Xia; Feng, Jianhua; Huang, Aiyou; He, Linwen; Niu, Jianfeng; Wang, Guangce

2017-11-01

Pyropia haitanensis has prominent stress-resistance characteristics and is endemic to China. Studies into the stress responses in these algae could provide valuable information on the stress-response mechanisms in the intertidal Rhodophyta. Here, the effects of salinity and light intensity on the quantum yield of photosystem II in Py. haitanensis were investigated using pulse-amplitude-modulation fluorometry. Total RNA and genomic DNA of the samples under different stress conditions were isolated. By normalizing to the genomic DNA quantity, the RNA content in each sample was evaluated. The cDNA was synthesized and the expression levels of seven potential internal control genes were evaluated using qRT-PCR method. Then, we used geNorm, a common statistical algorithm, to analyze the qRT-PCR data of seven reference genes. Potential genes that may constantly be expressed under different conditions were selected, and these genes showed stable expression levels in samples under a salinity treatment, while tubulin, glyceraldehyde-3-phosphate dehydrogenase and actin showed stability in samples stressed by strong light. Based on the results of the pulse amplitude-modulation fluorometry, an absolute quantification was performed to obtain gene copy numbers in certain stress-treated samples. The stably expressed genes as determined by the absolute quantification in certain samples conformed to the results of the geNorm screening. Based on the results of the software analysis and absolute quantification, we proposed that elongation factor 3 and 18S ribosomal RNA could be used as internal control genes when the Py. haitanensis blades were subjected to salinity stress, and that α-tubulin and 18S ribosomal RNA could be used as the internal control genes when the stress was from strong light. In general, our findings provide a convenient reference for the selection of internal control genes when designing experiments related to stress responses in Py. haitanensis.

Equipment qualification research program: program plan

International Nuclear Information System (INIS)

Dong, R.G.; Smith, P.D.

1982-01-01

The Lawrence Livermore National Laboratory (LLNL) under the sponsorship of the US Nuclear Regulatory Commission (NRC) has developed this program plan for research in equipment qualification (EQA). In this report the research program which will be executed in accordance with this plan will be referred to as the Equipment Qualification Research Program (EQRP). Covered are electrical and mechanical equipment under the conditions described in the OBJECTIVE section of this report. The EQRP has two phases; Phase I is primarily to produce early results and to develop information for Phase II. Phase I will last 18 months and consists of six projects. The first project is program management. The second project is responsible for in-depth evaluation and review of EQ issues and EQ processes. The third project is responsible for detailed planning to initiate Phase II. The remaining three projects address specific equipment; i.e., valves, electrical equipment, and a pump

40 CFR 68.175 - Prevention program/Program 3.

Science.gov (United States)

2010-07-01

... (CONTINUED) CHEMICAL ACCIDENT PREVENTION PROVISIONS Risk Management Plan § 68.175 Prevention program/Program 3. (a) For each Program 3 process, the owner or operator shall provide the information indicated in paragraphs (b) through (p) of this section. If the same information applies to more than one covered process...

A strategy for automatically generating programs in the lucid programming language

Science.gov (United States)

Johnson, Sally C.

1987-01-01

A strategy for automatically generating and verifying simple computer programs is described. The programs are specified by a precondition and a postcondition in predicate calculus. The programs generated are in the Lucid programming language, a high-level, data-flow language known for its attractive mathematical properties and ease of program verification. The Lucid programming is described, and the automatic program generation strategy is described and applied to several example problems.

Stochastic integer programming by dynamic programming

NARCIS (Netherlands)

Lageweg, B.J.; Lenstra, J.K.; Rinnooy Kan, A.H.G.; Stougie, L.; Ermoliev, Yu.; Wets, R.J.B.

1988-01-01

Stochastic integer programming is a suitable tool for modeling hierarchical decision situations with combinatorial features. In continuation of our work on the design and analysis of heuristics for such problems, we now try to find optimal solutions. Dynamic programming techniques can be used to

Applied Energy Program

Science.gov (United States)

Science Programs Applied Energy Programs Civilian Nuclear Energy Programs Laboratory Directed Research » Applied Energy Program Applied Energy Program Los Alamos is using its world-class scientific capabilities to enhance national energy security by developing energy sources with limited environmental impact

Computer-Assisted Program Reasoning Based on a Relational Semantics of Programs

Directory of Open Access Journals (Sweden)

Wolfgang Schreiner

2012-02-01

Full Text Available We present an approach to program reasoning which inserts between a program and its verification conditions an additional layer, the denotation of the program expressed in a declarative form. The program is first translated into its denotation from which subsequently the verification conditions are generated. However, even before (and independently of any verification attempt, one may investigate the denotation itself to get insight into the "semantic essence" of the program, in particular to see whether the denotation indeed gives reason to believe that the program has the expected behavior. Errors in the program and in the meta-information may thus be detected and fixed prior to actually performing the formal verification. More concretely, following the relational approach to program semantics, we model the effect of a program as a binary relation on program states. A formal calculus is devised to derive from a program a logic formula that describes this relation and is subject for inspection and manipulation. We have implemented this idea in a comprehensive form in the RISC ProgramExplorer, a new program reasoning environment for educational purposes which encompasses the previously developed RISC ProofNavigator as an interactive proving assistant.

Abstract Interpretation of PIC programs through Logic Programming

DEFF Research Database (Denmark)

Henriksen, Kim Steen; Gallagher, John Patrick

2006-01-01

, are applied to the logic based model of the machine. A small PIC microcontroller is used as a case study. An emulator for this microcontroller is written in Prolog, and standard programming transformations and analysis techniques are used to specialise this emulator with respect to a given PIC program....... The specialised emulator can now be further analysed to gain insight into the given program for the PIC microcontroller. The method describes a general framework for applying abstractions, illustrated here by linear constraints and convex hull analysis, to logic programs. Using these techniques on the specialised...

Playing by Programming: Making Gameplay a Programming Activity

Science.gov (United States)

Weintrop, David; Wilensky, Uri

2016-01-01

Video games are an oft-cited reason for young learners getting interested in programming and computer science. As such, many learning opportunities build on this interest by having kids program their own video games. This approach, while sometimes successful, has its drawbacks stemming from the fact that the challenge of programming and game…

40 CFR 68.170 - Prevention program/Program 2.

Science.gov (United States)

2010-07-01

... (CONTINUED) CHEMICAL ACCIDENT PREVENTION PROVISIONS Risk Management Plan § 68.170 Prevention program/Program 2. (a) For each Program 2 process, the owner or operator shall provide in the RMP the information... the process. (c) The name(s) of the chemical(s) covered. (d) The date of the most recent review or...

Verification of Imperative Programs by Constraint Logic Program Transformation

Directory of Open Access Journals (Sweden)

Emanuele De Angelis

2013-09-01

Full Text Available We present a method for verifying partial correctness properties of imperative programs that manipulate integers and arrays by using techniques based on the transformation of constraint logic programs (CLP. We use CLP as a metalanguage for representing imperative programs, their executions, and their properties. First, we encode the correctness of an imperative program, say prog, as the negation of a predicate 'incorrect' defined by a CLP program T. By construction, 'incorrect' holds in the least model of T if and only if the execution of prog from an initial configuration eventually halts in an error configuration. Then, we apply to program T a sequence of transformations that preserve its least model semantics. These transformations are based on well-known transformation rules, such as unfolding and folding, guided by suitable transformation strategies, such as specialization and generalization. The objective of the transformations is to derive a new CLP program TransfT where the predicate 'incorrect' is defined either by (i the fact 'incorrect.' (and in this case prog is not correct, or by (ii the empty set of clauses (and in this case prog is correct. In the case where we derive a CLP program such that neither (i nor (ii holds, we iterate the transformation. Since the problem is undecidable, this process may not terminate. We show through examples that our method can be applied in a rather systematic way, and is amenable to automation by transferring to the field of program verification many techniques developed in the field of program transformation.

Building Strong Geoscience Programs: Perspectives From Three New Programs

Science.gov (United States)

Flood, T. P.; Munk, L.; Anderson, S. W.

2005-12-01

During the past decade, at least sixteen geoscience departments in the U.S. that offer a B.S. degree or higher have been eliminated or dispersed. During that same time, three new geoscience departments with degree-granting programs have been developed. Each program has unique student demographics, affiliation (i.e. public institution versus private liberal arts college), geoscience curricula and reasons for initiation. Some of the common themes for each program include; 1) strong devotion to providing field experiences, 2) commitment to student-faculty collaborative research, 3) maintaining traditional geology program elements in the core curriculum and 4) placing students into high quality graduate programs and geoscience careers. Although the metrics for each school vary, each program can claim success in the area of maintaining solid enrollments. This metric is critical because programs are successful only if they have enough students, either in the major and/or general education courses, to convince administrators that continued support of faculty, including space and funding is warranted. Some perspectives gained through the establishment of these new programs may also be applicable to established programs. The success and personality of a program can be greatly affected by the personality of a single faculty member. Therefore, it may not be in the best interest of a program to distribute programmatic work equally among all faculty. For example, critical responsibilities such as teaching core and introductory courses should be the responsibility of faculty who are fully committed to these pursuits. However, if these responsibilities reduce scholarly output, well-articulated arguments should be developed in order to promote program quality and sustainability rather than individual productivity. Field and undergraduate research experiences should be valued as much as high-quality classroom and laboratory instruction. To gain the support of the administration

Interfacing ANSYS to user's programs using UNIX shell program

Energy Technology Data Exchange (ETDEWEB)

Kim, In Yong; Kim, Beom Shig [Korea Atomic Energy Research Institute, Taejon (Korea, Republic of)

1994-01-01

It has been considered to be impossible to interface the ANSYS, which is the commercial finite element code and whose program is not open to public, to the other user's program. When the analysis need to be iterated, the user should wait until the analysis is finished and read the ANSYS result to make the input data for every iteration. In this report the direct interfacing techniques between the ANSYS and the other program using UNIX shell programming are proposed. The detail program lists and the application example are also provided. (Author) 19 refs., 6 figs., 7 tabs.

Reference gene selection for quantitative real-time PCR analysis in virus infected cells: SARS corona virus, Yellow fever virus, Human Herpesvirus-6, Camelpox virus and Cytomegalovirus infections

Directory of Open Access Journals (Sweden)

Müller Marcel A

2005-02-01

Full Text Available Abstract Ten potential reference genes were compared for their use in experiments investigating cellular mRNA expression of virus infected cells. Human cell lines were infected with Cytomegalovirus, Human Herpesvirus-6, Camelpox virus, SARS coronavirus or Yellow fever virus. The expression levels of these genes and the viral replication were determined by real-time PCR. Genes were ranked by the BestKeeper tool, the GeNorm tool and by criteria we reported previously. Ranking lists of the genes tested were tool dependent. However, over all, β-actin is an unsuitable as reference gene, whereas TATA-Box binding protein and peptidyl-prolyl-isomerase A are stable reference genes for expression studies in virus infected cells.

Clean Coal Technology Demonstration Program: Program update 1993

Energy Technology Data Exchange (ETDEWEB)

1994-03-01

The Clean Coal Technology Demonstration Program (also referred to as the CCT Program) is a $6.9 billion cost-shared industry/government technology development effort. The program is to demonstrate a new generation of advanced coal-based technologies, with the most promising technologies being moved into the domestic and international marketplace. Technology has a vital role in ensuring that coal can continue to serve U.S. energy interests and enhance opportunities for economic growth and employment while meeting the national committment to a clean and healthy global environment. These technologies are being advanced through the CCT Program. The CCT Program supports three substantive national objectives: ensuring a sustainable environment through technology; enhancing energy efficiency and reliability; providing opportunities for economic growth and employment. The technologies being demonstrated under the CCT Program reduce the emissions of sulfur oxides, nitrogen oxides, greenhouse gases, hazardous air pollutants, solid and liquid wastes, and other emissions resulting from coal use or conversion to other fuel forms. These emissions reductions are achieved with efficiencies greater than or equal to currently available technologies.

75 FR 58468 - Terrorism Risk Insurance Program; Program Loss Reporting

Science.gov (United States)

2010-09-24

... DEPARTMENT OF THE TREASURY Terrorism Risk Insurance Program; Program Loss Reporting AGENCY: Departmental Offices, Terrorism Risk Insurance Program Office, Treasury. ACTION: Notice and request for... 1995, Public Law 104-13 (44 U.S.C. 3506(c)(2)(A)). Currently, the Terrorism Risk Insurance Program...

Functional Programming in C# Classic Programming Techniques for Modern Projects

CERN Document Server

Sturm, Oliver

2011-01-01

Take advantage of the growing trend in functional programming. C# is the number-one language used by .NET developers and one of the most popular programming languages in the world. It has many built-in functional programming features, but most are complex and little understood. With the shift to functional programming increasing at a rapid pace, you need to know how to leverage your existing skills to take advantage of this trend. Functional Programming in C# leads you along a path that begins with the historic value of functional ideas. Inside, C# MVP and functional programming expert Oli

PLC Programming

International Nuclear Information System (INIS)

Lee, Seong Jae; Wi, Seong Dong; Yoo, Jong Seon; Kim, Se Chan

2001-02-01

This book tells of PLC programming for KGL-WIN with summary of PLC, performance and function of PLC like characteristic of KGL-WIN, connection method with PLC, basic performance of K200S/K300S/K1000S, diagram of input and output H/W, writing project, staring the program, editing of program, on-line function, debugging and instructions like control, timer and counter, data transmission, comparison, rotation and moving, system, data operating data conversion and application program.

Material Programming

DEFF Research Database (Denmark)

Vallgårda, Anna; Boer, Laurens; Tsaknaki, Vasiliki

2017-01-01

. Consequently we ask what the practice of programming and giving form to such materials would be like? How would we be able to familiarize ourselves with the dynamics of these materials and their different combinations of cause and effect? Which tools would we need and what would they look like? Will we program......, and color, but additionally being capable of sensing, actuating, and computing. Indeed, computers will not be things in and by themselves, but embedded into the materials that make up our surroundings. This also means that the way we interact with computers and the way we program them, will change...... these computational composites through external computers and then transfer the code them, or will the programming happen closer to the materials? In this feature we outline a new research program that floats between imagined futures and the development of a material programming practice....

Practical C++ programming

National Research Council Canada - National Science Library

Oualline, Steve

2003-01-01

... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 4 6 3 2. The Basics of Program Writing Programs from Conception to Execution Creating a Real Program Getting Help in Unix Getting Help in an IDE Programming...

Clean Coal Technology Programs: Program Update 2009

Energy Technology Data Exchange (ETDEWEB)

None

2009-10-01

The purpose of the Clean Coal Technology Programs: Program Update 2009 is to provide an updated status of the U.S. Department of Energy (DOE) commercial-scale demonstrations of clean coal technologies (CCT). These demonstrations have been performed under the Clean Coal Technology Demonstration Program (CCTDP), the Power Plant Improvement Initiative (PPII), and the Clean Coal Power Initiative (CCPI). Program Update 2009 provides: (1) a discussion of the role of clean coal technology demonstrations in improving the nation’s energy security and reliability, while protecting the environment using the nation’s most abundant energy resource—coal; (2) a summary of the funding and costs of the demonstrations; and (3) an overview of the technologies being demonstrated, along with fact sheets for projects that are active, recently completed, or recently discontinued.

Program overview: Subsurface science program

International Nuclear Information System (INIS)

1994-03-01

The OHER Subsurface Science Program is DOE's core basic research program concerned with subsoils and groundwater. These practices have resulted in contamination by mixtures of organic chemicals, inorganic chemicals, and radionuclides. A primary long-term goal is to provide a foundation of knowledge that will lead to the reduction of environmental risks and to cost-effective cleanup strategies. Since the Program was initiated in 1985, a substantial amount of research in hydrogeology, subsurface microbiology, and the geochemistry of organically complexed radionuclides has been completed, leading to a better understanding of contaminant transport in groundwater and to new insights into microbial distribution and function in the subsurface environments. The Subsurface Science Program focuses on achieving long-term scientific advances that will assist DOE in the following key areas: providing the scientific basis for innovative in situ remediation technologies that are based on a concept of decontamination through benign manipulation of natural systems; understanding the complex mechanisms and process interactions that occur in the subsurface; determining the influence of chemical and geochemical-microbial processes on co-contaminant mobility to reduce environmental risks; improving predictions of contaminant transport that draw on fundamental knowledge of contaminant behavior in the presence of physical and chemical heterogeneities to improve cleanup effectiveness and to predict environmental risks

MatLab Programming for Engineers Having No Formal Programming Knowledge

Science.gov (United States)

Shaykhian, Linda H.; Shaykhian, Gholam Ali

2007-01-01

MatLab is one of the most widely used very high level programming languages for Scientific and engineering computations. It is very user-friendly and needs practically no formal programming knowledge. Presented here are MatLab programming aspects and not just the MatLab commands for scientists and engineers who do not have formal programming training and also have no significant time to spare for learning programming to solve their real world problems. Specifically provided are programs for visualization. Also, stated are the current limitations of the MatLab, which possibly can be taken care of by Mathworks Inc. in a future version to make MatLab more versatile.

C++ Programming Language

Science.gov (United States)

Shaykhian, Gholam Ali

2007-01-01

C++ Programming Language: The C++ seminar covers the fundamentals of C++ programming language. The C++ fundamentals are grouped into three parts where each part includes both concept and programming examples aimed at for hands-on practice. The first part covers the functional aspect of C++ programming language with emphasis on function parameters and efficient memory utilization. The second part covers the essential framework of C++ programming language, the object-oriented aspects. Information necessary to evaluate various features of object-oriented programming; including encapsulation, polymorphism and inheritance will be discussed. The last part of the seminar covers template and generic programming. Examples include both user defined and standard templates.

Site Support Program Plan Infrastructure Program

International Nuclear Information System (INIS)

1995-01-01

The Fiscal Year 1996 Infrastructure Program Site Support Program Plan addresses the mission objectives, workscope, work breakdown structures (WBS), management approach, and resource requirements for the Infrastructure Program. Attached to the plan are appendices that provide more detailed information associated with scope definition. The Hanford Site's infrastructure has served the Site for nearly 50 years during defense materials production. Now with the challenges of the new environmental cleanup mission, Hanford's infrastructure must meet current and future mission needs in a constrained budget environment, while complying with more stringent environmental, safety, and health regulations. The infrastructure requires upgrading, streamlining, and enhancement in order to successfully support the site mission of cleaning up the Site, research and development, and economic transition

Computer program CDCID: an automated quality control program using CDC update

International Nuclear Information System (INIS)

Singer, G.L.; Aguilar, F.

1984-04-01

A computer program, CDCID, has been developed in coordination with a quality control program to provide a highly automated method of documenting changes to computer codes at EG and G Idaho, Inc. The method uses the standard CDC UPDATE program in such a manner that updates and their associated documentation are easily made and retrieved in various formats. The method allows each card image of a source program to point to the document which describes it, who created the card, and when it was created. The method described is applicable to the quality control of computer programs in general. The computer program described is executable only on CDC computing systems, but the program could be modified and applied to any computing system with an adequate updating program

General edition program

International Nuclear Information System (INIS)

Vaturi, Sylvain

1969-01-01

Computerized edition is essential for data processing exploitation. When a more or less complex edition program is required for each task, then the need for a general edition program become obvious. The aim of this study is to create a general edition program. Universal programs are capable to execute numerous and varied tasks. For a more precise processing, the execution of which is frequently required, the use of a specialized program is preferable because, contradictory to the universal program, it goes straight to the point [fr

Preretirement Programs within Service Firms: Existing and Planned Programs.

Science.gov (United States)

Siegel, Sidney R.; Rives, Janet M.

1980-01-01

A study of 300 nonmanufacturing firms' current and projected preretirement programs indicated that (1) personnel departments have primary responsibility for existing programs, (2) focus is changing from financial planning to psychological counseling, and (3) such programs benefit the company as well as the employee. (SK)

Performance Demonstration Program Plan for the WIPP Experimental-Waste Characterization Program

International Nuclear Information System (INIS)

1991-02-01

The Performance Demonstration Program is designed to ensure that compliance with the Quality Assurance Objective, identified in the Quality Assurance Program Plan for the WIPP Experimental-Waste Characterization Program (QAPP), is achieved. This Program Plan is intended for use by the WPO to assess the laboratory support provided for the characterization of WIPP TRU waste by the storage/generator sites. Phase 0 of the Performance Demonstration Program encompasses the analysis of headspace gas samples for inorganic and organic components. The WPO will ensure the implementation of this plan by designating an independent organization to coordinate and provide technical oversight for the program (Program Coordinator). Initial program support, regarding the technical oversight and coordination functions, shall be provided by the USEPA-ORP. This plan identifies the criteria that will be used for the evaluation of laboratory performance, the responsibilities of the Program Coordinator, and the responsibilities of the participating laboratories. 5 tabs

Functional Python programming

CERN Document Server

Lott, Steven

2015-01-01

This book is for developers who want to use Python to write programs that lean heavily on functional programming design patterns. You should be comfortable with Python programming, but no knowledge of functional programming paradigms is needed.

Program evaluation: Weatherization Residential Assistance Partnership (WRAP) Program

Energy Technology Data Exchange (ETDEWEB)

1991-12-01

The Connecticut low income weatherization program was developed in response to a 1987 rate docket order from the Connecticut Department of Public Utility Control (DPUC) to Connecticut Light Power Co., an operating subsidiary of Northeast Utilities (NU). (Throughout this report, NU is referred to as the operator of the program.) This program, known as the Weatherization Residential Assistance Partnership, or WRAP, was configured utilizing input from a collaborative group of interested parties to the docket. It was agreed that this program would be put forth by the electric utility, but would not ignore oil and gas savings (thus, it was to be fuel- blind''). The allocated cost of conservation services for each fuel source, however, should be cost effective. It was to be offered to those utility customers at or below 200 percent of the federal poverty levels, and provide a wide array of energy saving measures directed toward heating, water heating and lighting. It was felt by the collaborative group that this program would raise the level of expenditures per participant for weatherization services provided by the state, and by linking to and revising the auditing process for weatherization, would lower the audit unit cost. The program plans ranged from the offering of low-cost heating, water heating and infiltration measures, increased insulation levels, carpentry and plumbing services, to furnace or burner replacement. The program was configured to allow for very comprehensive weatherization and heating system servicing.

Programming for controlling of pulse radiolysis setup. Program RADIO96

International Nuclear Information System (INIS)

Mirkowski, J.; Grodkowski, J.

1998-01-01

Program RADIO96 was written in Pascal using DELPHI 1.0 (Borland) programming platform. It can operate on IBM PC compatible computers in WINDOWS 3x or WINDOWS'95 environment. The program is dedicated to the pulse radiolysis setup based on the linear electron accelerator LAE 13/9 of the Department of Radiation Chemistry and Technology of the INCT. This work was based on apparatus and results described before and also on programming manuals of used equipment and technical data of programming platform. (author)

Programming F#

CERN Document Server

Smith, Chris

2009-01-01

Why learn F#? This multi-paradigm language not only offers you an enormous productivity boost through functional programming, it also lets you develop applications using your existing object-oriented and imperative programming skills. With Programming F#, you'll quickly discover the many advantages of Microsoft's new language, which includes access to all the great tools and libraries of the .NET platform. Learn how to reap the benefits of functional programming for your next project -- whether it's quantitative computing, large-scale data exploration, or even a pursuit of your own. With th

Programming Interactivity

CERN Document Server

Noble, Joshua

2009-01-01

Make cool stuff. If you're a designer or artist without a lot of programming experience, this book will teach you to work with 2D and 3D graphics, sound, physical interaction, and electronic circuitry to create all sorts of interesting and compelling experiences -- online and off. Programming Interactivity explains programming and electrical engineering basics, and introduces three freely available tools created specifically for artists and designers: Processing, a Java-based programming language and environment for building projects on the desktop, Web, or mobile phonesArduino, a system t

Choreographic Programming

DEFF Research Database (Denmark)

Montesi, Fabrizio

, as they offer a concise view of the message flows enacted by a system. For this reason, in the last decade choreographies have been used in the development of programming languages, giving rise to a programming paradigm that in this dissertation we refer to as Choreographic Programming. Recent studies show...... endpoint described in a choreography can then be automatically generated, ensuring that such implementations are safe by construction. However, current formal models for choreographies do not deal with critical aspects of distributed programming, such as asynchrony, mobility, modularity, and multiparty...... sessions; it remains thus unclear whether choreographies can still guarantee safety when dealing with such nontrivial features. This PhD dissertation argues for the suitability of choreographic programming as a paradigm for the development of safe distributed systems. We proceed by investigating its...

Teaching object-oriented programming on top of functional programming

DEFF Research Database (Denmark)

Kristensen, Jens Thyge; Hansen, Michael Reichhardt; Richel, Hans

2001-01-01

programming in the construction of OO programs. This is done following a method where the program design is expressed in SML and afterwards implemented in Java. The use of different languages in design and implementation is an advantage as it makes the distinction between these two stages very clear...

Site Support Program Plan Infrastructure Program

Energy Technology Data Exchange (ETDEWEB)

NONE

1995-09-26

The Fiscal Year 1996 Infrastructure Program Site Support Program Plan addresses the mission objectives, workscope, work breakdown structures (WBS), management approach, and resource requirements for the Infrastructure Program. Attached to the plan are appendices that provide more detailed information associated with scope definition. The Hanford Site`s infrastructure has served the Site for nearly 50 years during defense materials production. Now with the challenges of the new environmental cleanup mission, Hanford`s infrastructure must meet current and future mission needs in a constrained budget environment, while complying with more stringent environmental, safety, and health regulations. The infrastructure requires upgrading, streamlining, and enhancement in order to successfully support the site mission of cleaning up the Site, research and development, and economic transition.

Employee Assistance Programs.

Science.gov (United States)

Levine, Hermine Zagat

1985-01-01

The author reports company responses to a questionnaire concerning employee assistance programs (EAP). Answers concern EAP structure, staff training, use of outside consultant, services provided by EAPs, program administration, employee confidence in EAPs, advertising the program, program philosophy, problems encountered by EAP users, coverage and…

Assembly processor program converts symbolic programming language to machine language

Science.gov (United States)

Pelto, E. V.

1967-01-01

Assembly processor program converts symbolic programming language to machine language. This program translates symbolic codes into computer understandable instructions, assigns locations in storage for successive instructions, and computer locations from symbolic addresses.

75 FR 70083 - Emergency Forest Restoration Program and Emergency Conservation Program

Science.gov (United States)

2010-11-17

... Restoration Program and Emergency Conservation Program AGENCY: Farm Service Agency, USDA. ACTION: Interim rule. SUMMARY: The Farm Service Agency (FSA) is amending regulations as required by the Food, Conservation, and... as follows: PART 701--EMERGENCY CONSERVATION PROGRAM, EMERGENCY FOREST RESTORATION PROGRAM, AND...

Programming with joystick

OpenAIRE

Banič, Nejc

2014-01-01

In this thesis, we implemented a way of programming by means of gaming accessories. The main reason is that to show a diferent way of developing programs, because vast majority of programers are using two input / output devices: keyboard and mouse. These two devices have become standard and will definitely remain so in the future. For our implementation, we used high-level programming language Java and NetBeans integrated development environment. The program is actually a ...

Combining program visualization with programming workspace to assist students for completing programming laboratory task

Directory of Open Access Journals (Sweden)

Elvina Elvina

2018-06-01

Full Text Available Numerous Program Visualization tools (PVs have been developed for assisting novice students to understand their source code further. However, none of them are practical to be used in the context of completing programming laboratory task; students are required to keep switching between PV and programming workspace when they need to know how their code works. This paper combines PV with programming workspace to handle such issue. Resulted tool (which is named PITON has 13 features extracted from PythonTutor, PyCharm, and student’s feedbacks about PythonTutor. According to think-aloud and user study, PITON is more practical to be used than a combination of PythonTutor and PyCharm. Further, its features are considerably helpful; students rated these features as useful and frequently used.

An Attempt of Making Program-Generated Animation in a Beginners’ Programming Class

Science.gov (United States)

Matsuyama, Chieko; Nakashima, Toyoshiro; Ishii, Naohiro

In general, mathematical subjects are used for programming education in universities. In this case, many students lose the interest in the programming because the students have the preconception that is difficult to program by using the mathematical expressions. Especially beginners of the programming are a tendency to lose the interest. Therefore it is pointed out to use the subjects which do not need mathematical knowledge as much as possible. In this paper the authors have tried to make animation that are generated by programs instead of the mathematical subjects in a beginners’ programming class using C language used in a wide-ranging field. The authors discuss about improvements of the interest of students for programming by the try that is to make animation by programs in a programming class and refer to its effects.

Clean Coal Technology Programs: Program Update 2003 (Volume 1)

Energy Technology Data Exchange (ETDEWEB)

Assistant Secretary for Fossil Energy

2003-12-01

Annual report on the Clean Coal Technology Demonstration Program (CCTDP), Power Plant Improvement Initiative (PPII), and Clean Coal Power Initiative (CCPI). The report addresses the roles of the programs, implementation, funding and costs, project descriptions, legislative history, program history, environmental aspects, and project contacts. The project descriptions describe the technology and provides a brief summary of the demonstration results.

Active sites environmental monitoring Program - Program Plan: Revision 2

International Nuclear Information System (INIS)

Morrissey, C.M.; Hicks, D.S.; Ashwood, T.L.; Cunningham, G.R.

1994-05-01

The Active Sites Environmental Monitoring Program (ASEMP), initiated in 1989, provides early detection and performance monitoring of active low-level-waste (LLW) and transuranic (TRU) waste facilities at Oak Ridge National Laboratory (ORNL). Several changes have recently occurred in regard to the sites that are currently used for waste storage and disposal. These changes require a second set of revisions to the ASEMP program plan. This document incorporates those revisions. This program plan presents the organization and procedures for monitoring the active sites. The program plan also provides internal reporting levels to guide the evaluation of monitoring results

NASA Graduate Student Researchers Program Ronald E. McNair PhD Program

Science.gov (United States)

Howard, Sunnie

1998-01-01

The NASA Ronald E. McNair PHD Program was funded in September 1995. Implementation began during the spring of 1996. The deferment of the actual program initial semester enabled the program to continue support through the fall semester of 1998. This was accomplished by a no-cost extension from August 15, 1998 through December 31, 1998. There were 12 fellows supported by the program in 1996, 15 fellows in 1997, and 15 fellows 1998. Current program capacity is 15 fellows per funding support. Support for the academic outreach component began in spring 1998. The program was named the "Good Enough" Crew Activity (GECA) in honor of Dr. McNair's philosophy of everyone being good enough to achieve anything they want bad enough. The program currently enrolls 65 students from the third through the eight grades. The program is held 12 Saturdays per semester. The time is 9:00 AM to 12:30 PM each Saturday Morning. Program direction and facilitation is jointly administered with the PHD fellows and the Saturday Academy staff. Dr. John Kelly, REM-PHD Principal Investigator serves in a program oversight and leadership capacity. Ms. Sunnie Howard, The NASA REM-PHD Administrative Coordinator serves in an administrative and logistical capacity. Mr. Aaron Hatch, the NASA-AMES Liaison Officer, serve@'in a consultative and curriculum review capacity. The first recognition activity will be held on December 12, 1998, with the students, parents, faculty, PHD fellows, and other local student support services persons. Program outreach efforts are jointly supported by the NASA REM-PHD Program and the National Space Grant College and Fellowship Program. The Ph.D. program reached its first milestone in May 1998. North Carolina A&T State University graduated the first Ph.D. fellows. The first three Ph.D. Alumni were Ronald E. McNair PHD Program Fellows. It is hoped that this is just the beginning of a highly acclaimed doctoral program. The ultimate program success will be recognized when the

Implementing corporate wellness programs: a business approach to program planning.

Science.gov (United States)

Helmer, D C; Dunn, L M; Eaton, K; Macedonio, C; Lubritz, L

1995-11-01

1. Support of key decision makers is critical to the successful implementation of a corporate wellness program. Therefore, the program implementation plan must be communicated in a format and language readily understood by business people. 2. A business approach to corporate wellness program planning provides a standardized way to communicate the implementation plan. 3. A business approach incorporates the program planning components in a format that ranges from general to specific. This approach allows for flexibility and responsiveness to changes in program planning. 4. Components of the business approach are the executive summary, purpose, background, ground rules, approach, requirements, scope of work, schedule, and financials.

Cavity design programs

International Nuclear Information System (INIS)

Nelson, E.M.

1996-01-01

Numerous computer programs are available to help accelerator physicists and engineers model and design accelerator cavities and other microwave components. This article discusses the problems these programs solve and the principles upon which these programs are based. Some examples of how these programs are used in the design of accelerator cavities are also given

Employee assistance programs: history and program description.

Science.gov (United States)

Gilbert, B

1994-10-01

1. The history and development of Employee Assistance Programs (EAPs) can be traced back to the 1800s. There are currently over 10,000 EAPs in the United States. 2. Standards for program accreditation and counselor certification have been established for EAPs. The "core technology of Employee Assistance Programs" includes identification of behavioural problems based on job performance issues, expert consultation with supervisors, appropriate use of constructive confrontation, microlinkages with treatment providers and resources, macrolinkages between providers, resources, and work organizations, focus on substance abuse, and evaluation of employee success based on job performance. 3. Some EAPs take a broad brush approach, and incorporate health promotion and managed care functions.

FY85 Program plan for the Defense Transuranic Waste Program (DTWP)

International Nuclear Information System (INIS)

1984-11-01

The Defense TRU Waste Program (DTWP) is the focal point for the Department of Energy in national planning, integration, and technical development for TRU waste management. The scope of this program extends from the point of TRU waste generation through delivery to a permanent repository. The TRU program maintains a close interface with repository development to ensure program compatibility and coordination. The defense TRU program does not directly address commercial activities that generate TRU waste. Instead, it is concerned with providing alternatives to manage existing and future defense TRU wastes. The FY85 Program Plan is consistent with the Defense TRU Waste Program goals and objectives stated in the Defense Transuranic Waste Program Strategy Document, January 1984. The roles of participants, the responsibilities and authorities for Research and Development (R and D), the organizational interfaces and communication channels for R and D and the establishment of procedures for planning, reporting, and budgeting of all R and D activities meet requirements stated in the Technical Management Plan for the Transuranic Waste Management Program. The Program Plan is revised as needed. The work breakdown structure is reflected graphically immediately following the Administration section and is described in the subsequent narrative. Detailed budget planning (i.e., programmatic funding and capital equipment) is presented for FY85; outyear budget projections are presented for future years

An Intelligent Robot Programing

Energy Technology Data Exchange (ETDEWEB)

Hong, Seong Yong

2012-01-15

This book introduces an intelligent robot programing with background of the begging, introduction of VPL, and SPL, building of environment for robot platform, starting of robot programing, design of simulation environment, robot autonomy drive control programing, simulation graphic. Such as SPL graphic programing graphical image and graphical shapes, and graphical method application, application of procedure for robot control, robot multiprogramming, robot bumper sensor programing, robot LRF sencor programing and robot color sensor programing.

Purely Functional Structured Programming

OpenAIRE

Obua, Steven

2010-01-01

The idea of functional programming has played a big role in shaping today's landscape of mainstream programming languages. Another concept that dominates the current programming style is Dijkstra's structured programming. Both concepts have been successfully married, for example in the programming language Scala. This paper proposes how the same can be achieved for structured programming and PURELY functional programming via the notion of LINEAR SCOPE. One advantage of this proposal is that m...

An Intelligent Robot Programing

International Nuclear Information System (INIS)

Hong, Seong Yong

2012-01-01

This book introduces an intelligent robot programing with background of the begging, introduction of VPL, and SPL, building of environment for robot platform, starting of robot programing, design of simulation environment, robot autonomy drive control programing, simulation graphic. Such as SPL graphic programing graphical image and graphical shapes, and graphical method application, application of procedure for robot control, robot multiprogramming, robot bumper sensor programing, robot LRF sencor programing and robot color sensor programing.

Multi-dimensional Rankings, Program Termination, and Complexity Bounds of Flowchart Programs

OpenAIRE

Alias , Christophe; Darte , Alain; Feautrier , Paul; Gonnord , Laure

2010-01-01

International audience; Proving the termination of a flowchart program can be done by exhibiting a ranking function, i.e., a function from the program states to a well-founded set, which strictly decreases at each program step. A standard method to automatically generate such a function is to compute invariants for each program point and to search for a ranking in a restricted class of functions that can be handled with linear programming techniques. Previous algorithms based on affine rankin...

Programming in COBOL

CERN Document Server

Lancaster, G T

2014-01-01

Programming in COBOL is a simple yet concise how-to book that teaches the programming language in a short yet effective step-by-step manner, which can be easily understood by anyone with sufficient knowledge in information technology. Covering first the advantages of COBOL over other programming languages, the book discusses COBOL's divisions - identification, environment, procedure, and data, and then describes the testing of the COBOL source programs and program questions. The book is valuable for those who wish to learn basic COBOL language, but do not have the time to take manufacturers' o

Fusion Energy Postdoctoral Research Program, Professional Development Program: FY 1987 annual report

International Nuclear Information System (INIS)

1988-01-01

In FY 1986, Oak Ridge Associated Universities (ORAU) initiated two programs for the US Department of Energy (DOE), Office of Fusion Energy (OFE): the Fusion Energy Postdoctoral Research Program and the Fusion Energy Professional Development Program. These programs provide opportunities to conduct collaborative research in magnetic fusion energy research and development programs at DOE laboratories and contractor sites. Participants become trained in advanced fusion energy research, interact with outstanding professionals, and become familiar with energy-related national issues while making personal contributions to the search for solutions to scientific problems. Both programs enhance the national fusion energy research and development effort by providing channels for the exchange of scientists and engineers, the diffusion of ideas and knowledge, and the transfer of relevant technologies. These programs, along with the Magnetic Fusion Energy Science and Technology Fellowship Programs, compose the fusion energy manpower development programs administered by ORAU for DOE/OFE

Program Fullerene

DEFF Research Database (Denmark)

Wirz, Lukas; Peter, Schwerdtfeger,; Avery, James Emil

2013-01-01

Fullerene (Version 4.4), is a general purpose open-source program that can generate any fullerene isomer, perform topological and graph theoretical analysis, as well as calculate a number of physical and chemical properties. The program creates symmetric planar drawings of the fullerene graph, an......-Fowler, and Brinkmann-Fowler vertex insertions. The program is written in standard Fortran and C++, and can easily be installed on a Linux or UNIX environment....

SPOT Program

Science.gov (United States)

Smith, Jason T.; Welsh, Sam J.; Farinetti, Antonio L.; Wegner, Tim; Blakeslee, James; Deboeck, Toni F.; Dyer, Daniel; Corley, Bryan M.; Ollivierre, Jarmaine; Kramer, Leonard;

Effectiveness and Successful Program Elements of SOAR’s Afterschool Programs

Directory of Open Access Journals (Sweden)

Janet L. Johnson

2007-03-01

Full Text Available Project SOAR provided after-school programs that afforded expanded learning opportunities to help students succeed in local public schools and to contribute to the general welfare of the community. Program components focused on building students’ academic skills and positive attitudes, aided by teachers, mentors, parent education, and local agencies. Instructional programs were conducted to help reduce drug use and violence. Activities included academic assistance, technology training, mentoring, service learning projects, and education in life skills and the arts. Parent involvement was encouraged. Behavioral and academic outcomes—especially at the high school level—were analyzed to determine program effectiveness regarding academic achievement, dropout rates, and rates and frequency of suspensions. Successful program elements and strategies are noted.

Nuclear Power Infrastructure Development Program: Korean Education Program

International Nuclear Information System (INIS)

Choi, Sung Yeol; Hwang, Il Soon; Kim, Si Hwan

2009-01-01

Many countries have decided nuclear power for next energy resources as one of the long-term energy supply options. IAEA projected nuclear power expansion up to 2030 reaching between 447 GWe and 691 GWe compared to 370 GWe and 2660 TWh at the end of 2006. Both low and high projection is accompanied with new nuclear power plant constructions respectively 178 and 357, about 11 units per year, and most new construction is in North America, the Far East, Eastern Europe, the Middle East, and Southeast Asia. During the last forty years, thirty three countries have established commercial nuclear power programs but only some of them have developed comprehensive and large scale peaceful nuclear power infrastructure. Although various cooperation and guidance program of nuclear power infrastructure, developing appropriate environment and infrastructure of nuclear power plant is still challenging problems for developing countries launching nuclear power program. With increasing the demand of safety and safeguard from international society, creating appropriate infrastructure becomes essential requirements in national nuclear power program. In the viewpoint of developing countries, without sufficient explanation and proper guidance, infrastructure could be seen only as another barrier in its nuclear power program. The importance of infrastructure development would be obscured by ostensible business and infrastructure program can result in increasing entering barriers to peaceful nuclear power application field without benefits to developing countries and international community. To avoid this situation by providing enough explanation and realistic case example and cooperate with the countries wanting to establish comprehensive nuclear power infrastructure in the peaceful applications, we are creating the education program of infrastructure development with basic guidelines of the IAEA infrastructure series and Korean experiences from least developed country to advanced country

NEK AOV program

International Nuclear Information System (INIS)

Fable, D.; Andric, A.; Prah, M.; Habinc, M.

2002-01-01

Air Operated Valves (AOVs) are used in a wide variety of applications in nuclear power plants. Some AOVs perform important functions in safety and non-safety related systems which could affect initiating event frequencies, accident mitigation and radiological releases. A review of lessons learned from nuclear plant MOV programs and pilot AOV programs indicates that AOV performance can be enhanced via improvements in valve and actuator sizing, setup, testing and maintenance. Nuklearna elektrarna Krsko has decided to develop an AOV program on the basis of Joint Owners. Group (JOG) AOV Program, which identifies nine key elements for successful implementation. The first step in establishing an AOV Program is to identify and categorize the plant AOVs for evaluation. Performed categorization in NEK has resulted with Preliminary AOV Scope. Important part of NEK AOV Program is NEK AOV Database. For the time being, basic structure with tables, queries, forms, reports and relationships has been created. Further development will be coordinated with development of NEK AOV Program Methodology.(author)

Utility green pricing programs: a statistical analysis of program effectiveness

International Nuclear Information System (INIS)

Ryan, W.; Scott, O.; Lori, B.; Blair, S.

2005-01-01

Utility green pricing programs represent one way in which consumers can voluntarily support the development of renewable energy. The design features and effectiveness of these programs varies considerably. Based on a survey of utility program managers in the United States, this article provides insight into which program features might help maximize both customer participation in green pricing programs and the amount of renewable energy purchased by customers in those programs. We find that program length has a substantial impact on customer participation and purchases; to achieve higher levels of success, utilities will need to remain committed to their product offering for some time. Our findings also suggest that utilities should consider higher renewable energy purchase thresholds for residential customers in order to maximize renewable energy sales. Smaller utilities are found to be more successful than larger utilities, and we find some evidence that providing private benefits to nonresidential participants can enhance success. Interestingly, we find little evidence that the cost of the green pricing product greatly impacts customer participation and renewable energy sales, at least over the narrow range of premiums embedded in our data set, and for the initial set of green power purchasers. (author)

The Y2K program for scientific-analysis computer programs at AECL

International Nuclear Information System (INIS)

Popovic, J.; Gaver, C.; Chapman, D.

1999-01-01

The evaluation of scientific-analysis computer programs for year-2000 compliance is part of AECL' s year-2000 (Y2K) initiative, which addresses both the infrastructure systems at AECL and AECL's products and services. This paper describes the Y2K-compliance program for scientific-analysis computer codes. This program involves the integrated evaluation of the computer hardware, middleware, and third-party software in addition to the scientific codes developed in-house. The project involves several steps: the assessment of the scientific computer programs for Y2K compliance, performing any required corrective actions, porting the programs to Y2K-compliant platforms, and verification of the programs after porting. Some programs or program versions, deemed no longer required in the year 2000 and beyond, will be retired and archived. (author)

The Y2K program for scientific-analysis computer programs at AECL

International Nuclear Information System (INIS)

Popovic, J.; Gaver, C.; Chapman, D.

1999-01-01

The evaluation of scientific analysis computer programs for year-2000 compliance is part of AECL's year-2000 (Y2K) initiative, which addresses both the infrastructure systems at AECL and AECL's products and services. This paper describes the Y2K-compliance program for scientific-analysis computer codes. This program involves the integrated evaluation of the computer hardware, middleware, and third-party software in addition to the scientific codes developed in-house. The project involves several steps: the assessment of the scientific computer programs for Y2K compliance, performing any required corrective actions, porting the programs to Y2K-compliant platforms, and verification of the programs after porting. Some programs or program versions, deemed no longer required in the year 2000 and beyond, will be retired and archived. (author)

Effective Programming

DEFF Research Database (Denmark)

Frost, Jacob

To investigate the use of VTLoE as a basis for formal derivation of functional programs with effects. As a part of the process, a number of issues central to effective formal programming are considered. In particular it is considered how to develop a proof system suitable for pratical reasoning......, how to implement this system in the generic proof assistant Isabelle and finally how to apply the logic and the implementation to programming....

Transgenerational developmental programming.

Science.gov (United States)

Aiken, Catherine E; Ozanne, Susan E

2014-01-01

The concept of developmental programming suggests that the early life environment influences offspring characteristics in later life, including the propensity to develop diseases such as the metabolic syndrome. There is now growing evidence that the effects of developmental programming may also manifest in further generations without further suboptimal exposure. This review considers the evidence, primarily from rodent models, for effects persisting to subsequent generations, and evaluates the mechanisms by which developmental programming may be transmitted to further generations. In particular, we focus on the potential role of the intrauterine environment in contributing to a developmentally programmed phenotype in subsequent generations. The literature was systematically searched at http://pubmed.org and http://scholar.google.com to identify published findings regarding transgenerational (F2 and beyond) developmental programming effects in human populations and animal models. Transmission of programming effects is often viewed as a form of epigenetic inheritance, either via the maternal or paternal line. Evidence exists for both germline and somatic inheritance of epigenetic modifications which may be responsible for phenotypic changes in further generations. However, there is increasing evidence for the role of both extra-genomic components of the zygote and the interaction of the developing conceptus with the intrauterine environment in propagating programming effects. The contribution of a suboptimal reproductive tract environment or maternal adaptations to pregnancy may be critical to inheritance of programming effects via the maternal line. As the effects of age exacerbate the programmed metabolic phenotype, advancing maternal age may increase the likelihood of developmental programming effects being transmitted to further generations. We suggest that developmental programming effects could be propagated through the maternal line de novo in generations

An Analysis on Distance Education Computer Programming Students' Attitudes Regarding Programming and Their Self-Efficacy for Programming

Science.gov (United States)

Ozyurt, Ozcan

2015-01-01

This study aims to analyze the attitudes of students studying computer programming through the distance education regarding programming, and their self-efficacy for programming and the relation between these two factors. The study is conducted with 104 students being thought with distance education in a university in the north region of Turkey in…

Mixed language programming

International Nuclear Information System (INIS)

Burow, Burkhard D.

1996-01-01

Computing in the next millennium will be using software from this millennium. Programming languages evolve and new ones continue to be created. The use of legacy code demonstrates why some present and future applications may span programming languages. Even a completely new application may mix programming languages, if it allows its components to be more conveniently expressed. Given the need, mixed language programming should be easy and robust. By resolving a variety of difficulties, the well established cfortran.h package provides, the desired convenient interface across the C and Fortran programming languages, as demonstrated using CERN's Book. (author)

Short rotation woody crops program: 1982 program summary

Energy Technology Data Exchange (ETDEWEB)

Ranney, J.W.; Cushman, J.H.; Trimble, J.L.

1982-09-01

The general objective and overall approach of the program are discussed, and the status, specific goals, and recent results of the three major research areas are presented. The report concludes with brief summaries of the individual research projects in the program.

Analysis of the Education Program Approval Process: A Program Evaluation.

Science.gov (United States)

Fountaine, Charles A.; And Others

A study of the education program approval process involving the Veterans Administration (VA) and the State Approving Agencies (SAAs) had the following objectives: to describe the present education program approval process; to determine time and costs associated with the education program approval process; to describe the approval process at…

14 CFR 91.1411 - Continuous airworthiness maintenance program use by fractional ownership program manager.

Science.gov (United States)

2010-01-01

... program use by fractional ownership program manager. 91.1411 Section 91.1411 Aeronautics and Space FEDERAL... airworthiness maintenance program use by fractional ownership program manager. Fractional ownership program... through 91.1443. Any program manager who elects to maintain the program aircraft using a continuous...

Program reference schedule baseline

International Nuclear Information System (INIS)

1986-07-01

This Program Reference Schedule Baseline (PRSB) provides the baseline Program-level milestones and associated schedules for the Civilian Radioactive Waste Management Program. It integrates all Program-level schedule-related activities. This schedule baseline will be used by the Director, Office of Civilian Radioactive Waste Management (OCRWM), and his staff to monitor compliance with Program objectives. Chapter 1 includes brief discussions concerning the relationship of the PRSB to the Program Reference Cost Baseline (PRCB), the Mission Plan, the Project Decision Schedule, the Total System Life Cycle Cost report, the Program Management Information System report, the Program Milestone Review, annual budget preparation, and system element plans. Chapter 2 includes the identification of all Level 0, or Program-level, milestones, while Chapter 3 presents and discusses the critical path schedules that correspond to those Level 0 milestones

Recommended programming practices to facilitate the portability of science computer programs

International Nuclear Information System (INIS)

Anon.

1983-01-01

This standard recommends programming practices to facilitate the portability of computer programs prepared for scientific and engineering computations. These practices are intended to simplify implementation, conversion, and modification of computer programs

78 FR 40084 - Proposed Requirement-Migrant Education Program Consortium Incentive Grant Program

Science.gov (United States)

2013-07-03

... DEPARTMENT OF EDUCATION 34 CFR Chapter II Proposed Requirement--Migrant Education Program... educational agencies (SEAs) under the Migrant Education Program (MEP) Consortium Incentive Grant (CIG) Program... the interstate or intrastate coordination of migrant education programs by addressing key needs of...

Medical student education program in Alzheimer’s disease: The PAIRS Program

Directory of Open Access Journals (Sweden)

Jefferson Angela L

2012-08-01

Full Text Available Abstract Background As life expectancy increases, dementia incidence will also increase, creating a greater need for physicians well-trained to provide integrated geriatric care. However, research suggests medical students have limited knowledge or interest in pursuing geriatric or dementia care. The purpose of this study is to evaluate the PAIRS Program and its effectiveness in enhancing medical education as a service-learning activity and replication model for the Buddy ProgramTM. Methods Between 2007 and 2011, four consecutive classes of first year Boston University School of Medicine students (n = 45; 24 ± 3 years, 58% female, 53% White participated in a year-long program in which they were paired with a patient with early-stage Alzheimer’s disease (AD. Assessments included pre- and post-program dementia knowledge tests and a post-program reflective essay. Results Program completion was 100% (n = 45. A paired-sample t-test revealed a modest improvement in dementia knowledge post-program (p  Conclusions Quantitative and qualitative findings suggest that the PAIRS Program can enhance the acquisition of knowledge, skills, and positive attitudes regarding geriatric healthcare in future generations of physicians, a skill set that is becoming increasingly relevant in light of the rapidly aging population. Furthermore, results suggest that The Buddy ProgramTM model can be successfully replicated.

Evaluation of the Health Rocks! Program: The Association of Youth Engagement with Program Outcomes

Directory of Open Access Journals (Sweden)

Yan Xia

2017-01-01

Full Text Available This evaluation research examined the relationship between program process and program outcome, specifically, youth engagement in the national 4-H Council Health Rocks! program and their program outcomes.  Based on program evaluation surveys completed after the program by participants, youths’ engagement in the program was associated with their gains in knowledge and skills about substance use, and personal assets related to avoiding risks.  When youth participants find a program interesting, are actively engaged in the program, and find the program staff friendly, they benefit more from the program.  Findings underscore the importance of engaging curriculum and friendly staff to the success of extension or afterschool youth programs. The evaluation method may offer an example of balancing rigor of evaluation design and feasibility of implementing an evaluation.

Research Program Overview

Science.gov (United States)

PEER logo Pacific Earthquake Engineering Research Center home about peer news events research products laboratories publications nisee b.i.p. members education FAQs links research Research Program Overview Tall Buildings Initiative Transportation Research Program Lifelines Program Concrete Grand

Programming Guidelines for FBD Programs in Reactor Protection System Software

International Nuclear Information System (INIS)

Jung, Se Jin; Lee, Dong Ah; Kim, Eui Sub; Yoo, Jun Beom; Lee, Jang Su

2014-01-01

Properties of programming languages, such as reliability, traceability, etc., play important roles in software development to improve safety. Several researches are proposed guidelines about programming to increase the dependability of software which is developed for safety critical systems. Misra-c is a widely accepted programming guidelines for the C language especially in the sector of vehicle industry. NUREG/CR-6463 helps engineers in nuclear industry develop software in nuclear power plant systems more dependably. FBD (Function Block Diagram), which is one of programming languages defined in IEC 61131-3 standard, is often used for software development of PLC (programmable logic controllers) in nuclear power plants. Software development for critical systems using FBD needs strict guidelines, because FBD is a general language and has easily mistakable elements. There are researches about guidelines for IEC 61131-3 programming languages. They, however, do not specify details about how to use languages. This paper proposes new guidelines for the FBD based on NUREG/CR-6463. The paper introduces a CASE (Computer-Aided Software Engineering) tool to check FBD programs with the new guidelines and shows availability with a case study using a FBD program in a reactor protection system. The paper is organized as follows

Programming Guidelines for FBD Programs in Reactor Protection System Software

Energy Technology Data Exchange (ETDEWEB)

Jung, Se Jin; Lee, Dong Ah; Kim, Eui Sub; Yoo, Jun Beom [Division of Computer Science and Engineering College of Information and Communication, Konkuk University, Seoul (Korea, Republic of); Lee, Jang Su [Man-Machine Interface System team Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

2014-10-15

Properties of programming languages, such as reliability, traceability, etc., play important roles in software development to improve safety. Several researches are proposed guidelines about programming to increase the dependability of software which is developed for safety critical systems. Misra-c is a widely accepted programming guidelines for the C language especially in the sector of vehicle industry. NUREG/CR-6463 helps engineers in nuclear industry develop software in nuclear power plant systems more dependably. FBD (Function Block Diagram), which is one of programming languages defined in IEC 61131-3 standard, is often used for software development of PLC (programmable logic controllers) in nuclear power plants. Software development for critical systems using FBD needs strict guidelines, because FBD is a general language and has easily mistakable elements. There are researches about guidelines for IEC 61131-3 programming languages. They, however, do not specify details about how to use languages. This paper proposes new guidelines for the FBD based on NUREG/CR-6463. The paper introduces a CASE (Computer-Aided Software Engineering) tool to check FBD programs with the new guidelines and shows availability with a case study using a FBD program in a reactor protection system. The paper is organized as follows.

IMPACT OF THE U.S. SUPPORT PROGRAM SAFEGUARDS INTERNSHIP PROGRAM

International Nuclear Information System (INIS)

PEPPER, S.; OSIECKI, C.

2006-01-01

The U.S. Support Program began funding an internship program in the IAEA Department of Safeguards in 2002. Since that time, 39 U.S. citizens and permanent residents have been placed in one-year, paid internships with the IAEA. The management of the internship program was originally the responsibility of the International Safeguards Project Office but was transferred to the Office of Educational Programs at Brookhaven National Laboratory in 2004. Feedback on the internship program from the U.S. government and the IAEA has been positive. The interns have completed basic yet essential work for the Department of Safeguards and freed IAEA staff members to perform more complex tasks. The cost of an intern is low relative to other forms of human resources support. After the conclusion of their assignments, many of the interns go on to work for the U.S. government, the national laboratories, or companies in international safeguards and nonproliferation. This paper will discuss the work done by the interns for the IAEA, factors influencing the success of the internship program, and the effects the program has had on the careers of interns, in preparing the next generation to work in the nuclear industry, participation in INMM activities, and recruitment for U.S. citizens for safeguards positions

Clean Coal Technology Demonstration Program. Program update 1994

Energy Technology Data Exchange (ETDEWEB)

NONE

1995-04-01

The Clean Coal Technology Demonstration Program (CCT Program) is a $7.14 billion cost-shared industry/government technology development effort. The program is to demonstrate a new generation of advanced coal-based technologies, with the most promising technologies being moved into the domestic and international marketplace. Clean coal technologies being demonstrated under the CCT program are creating the technology base that allows the nation to meet its energy and environmental goals efficiently and reliably. The fact that most of the demonstrations are being conducted at commercial scale, in actual user environments, and under conditions typical of commercial operations allows the potential of the technologies to be evaluated in their intended commercial applications. The technologies are categorized into four market sectors: advanced electric power generation systems; environmental control devices; coal processing equipment for clean fuels; and industrial technologies. Sections of this report describe the following: Role of the Program; Program implementation; Funding and costs; The road to commercial realization; Results from completed projects; Results and accomplishments from ongoing projects; and Project fact sheets. Projects include fluidized-bed combustion, integrated gasification combined-cycle power plants, advanced combustion and heat engines, nitrogen oxide control technologies, sulfur dioxide control technologies, combined SO{sub 2} and NO{sub x} technologies, coal preparation techniques, mild gasification, and indirect liquefaction. Industrial applications include injection systems for blast furnaces, coke oven gas cleaning systems, power generation from coal/ore reduction, a cyclone combustor with S, N, and ash control, cement kiln flue gas scrubber, and pulse combustion for steam coal gasification.

75 FR 67751 - Medicare Program: Community-Based Care Transitions Program (CCTP) Meeting

Science.gov (United States)

2010-11-03

...] Medicare Program: Community-Based Care Transitions Program (CCTP) Meeting AGENCY: Centers for Medicare... guidance and ask questions about the upcoming Community-based Care Transitions Program. The meeting is open... conference will also provide an overview of the Community-based Care Transitions Program (CCTP) and provide...

SSCL quality program overview

International Nuclear Information System (INIS)

Hedderick, R.V.; Threatt, D.C.

1992-01-01

The Quality Program for the Superconducing Super Collider Laboratory (SSCL) was developed for a number of reasons. The need for a quality program not only is a contractual requirement, but it also makes good economic sense to implement such a program. The quality program is the device used to coordinate the activities of different Laboratory organizations, such as Engineering and Procurement, and to improve operational reliability and safety. To be successful, the QA Program not only must satisfy Department of Energy (DOE) requirements and provide for flowdown of requirements to performing organizations, but must also be flexible enough so that the program is tailored to meet the needs of each internal organization. The keys to success are management support, acceptance by personnel, and cost effectiveness. These three items are assured by involving appropriate management at each step of program development, by personnel training and by feedback, and by programs to reduce defects and improve quality. Equally valuable is involvement of key organizations in program development. We will describe the basic SSCL Quality Program requirements, how the requirements are tailored to the needs of Laboratory organizations, and how the effectiveness of the program is validated

UNDERSTANDING THE RELATIONSHIPS OF PROGRAM SATISFACTION, PROGRAM LOYALTY AND STORE LOYALTY AMONG CARDHOLDERS OF LOYALTY PROGRAMS

OpenAIRE

Nor Asiah Omar; Norzalita Abd. Aziz; Muhamad Azrin Nazri

2011-01-01

Loyalty programs have increasingly attracted interest in both academic marketing research and practice. One major factor that has been increasingly discussed is loyalty. In this study we examine the influence of cardholders' satisfaction on loyalty (program loyalty and store loyalty) in a retail context, namely, in department stores and superstores. Data were collected from 400 cardholders of a retail loyalty program in Klang Valley, Malaysia via the drop-off-and-collect technique. Structural...

Icobj Programming

OpenAIRE

Boussinot , Frédéric

1996-01-01

A simple and fully graphical programming method is presented, using a powerful means to combine behaviors. This programming is based on the notion of an «icobj» which has a behavioral aspect («object» part), a graphical aspect («icon» part), with an «animation» aspect. Icobj programming provides parallelism, broadcast event communication and migration through the network. An experimental system based on this approach is described in details. Its implementation with reactive scripts is also pr...

Ostomy Home Skills Program

Medline Plus

Full Text Available ... Membership Directory 2017 Annual Meeting 2016 Annual Meeting Women's Committee Mentorship Program Outside Activities ACS Archives Contact Us Quality Programs Quality Programs Overview About Quality Programs ACS ...

The Army Preposition Afloat Program: Is It a Program We Need?

National Research Council Canada - National Science Library

Curl, William

1998-01-01

... of most required classes. This paper addresses the evolution of the APA program, describes the current and planned APA program, discusses management of the APA, and looks critically at the value of the program in support...

RIPE [robot independent programming environment]: A robot independent programming environment

International Nuclear Information System (INIS)

Miller, D.J.; Lennox, R.C.

1990-01-01

Remote manual operations in radiation environments are typically performed very slowly. Sensor-based computer-controlled robots hold great promise for increasing the speed and safety of remote operations; however, the programming of robotic systems has proven to be expensive and difficult. Generalized approaches to robot programming that reuse available software modules and employ programming languages which are independent of the specific robotic and sensory devices being used are needed to speed software development and increase overall system reliability. This paper discusses the robot independent programming environment (RIPE) developed at Sandia National Laboratories (SNL). The RIPE is an object-oriented approach to robot system architectures; it is a software environment that facilitates rapid design and implementation of complex robot systems for diverse applications. An architecture based on hierarchies of distributed multiprocessors provides the computing platform for a layered programming structure that models applications using software objects. These objects are designed to support model-based automated programming of robotic and machining devices, real-time sensor-based control, error handling, and robust communication

76 FR 27986 - Funding for the Conservation Loan Program; Farm Loan Programs

Science.gov (United States)

2011-05-13

... for the Conservation Loan Program; Farm Loan Programs AGENCY: Farm Service Agency, USDA. ACTION... or guaranteed loan applications for the Conservation Loan (CL) Program because of lack of program... provisions to the existing direct and guaranteed loan regulations found in 7 CFR parts 761, 762, 764, 765...

Programming Models in HPC

Energy Technology Data Exchange (ETDEWEB)

Shipman, Galen M. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

2016-06-13

These are the slides for a presentation on programming models in HPC, at the Los Alamos National Laboratory's Parallel Computing Summer School. The following topics are covered: Flynn's Taxonomy of computer architectures; single instruction single data; single instruction multiple data; multiple instruction multiple data; address space organization; definition of Trinity (Intel Xeon-Phi is a MIMD architecture); single program multiple data; multiple program multiple data; ExMatEx workflow overview; definition of a programming model, programming languages, runtime systems; programming model and environments; MPI (Message Passing Interface); OpenMP; Kokkos (Performance Portable Thread-Parallel Programming Model); Kokkos abstractions, patterns, policies, and spaces; RAJA, a systematic approach to node-level portability and tuning; overview of the Legion Programming Model; mapping tasks and data to hardware resources; interoperability: supporting task-level models; Legion S3D execution and performance details; workflow, integration of external resources into the programming model.

Generating Consistent Program Tutorials

DEFF Research Database (Denmark)

Vestdam, Thomas

2002-01-01

In this paper we present a tool that supports construction of program tutorials. A program tutorial provides the reader with an understanding of an example program by interleaving fragments of source code and explaining text. An example program can for example illustrate how to use a library or a......, and we see potential in using the tool to produce program tutorials to be used for frameworks, libraries, and in educational contexts.......In this paper we present a tool that supports construction of program tutorials. A program tutorial provides the reader with an understanding of an example program by interleaving fragments of source code and explaining text. An example program can for example illustrate how to use a library...... or a framework. We present a means for specifying the fragments of a program that are to be in-lined in the tutorial text. These in-line fragments are defined by addressing named syntactical elements, such as classes and methods, but it is also possible to address individual code lines by labeling them...

Program History

Science.gov (United States)

Learn how the National Cancer Institute transitioned the former Cooperative Groups Program to the National Clinical Trials Network (NCTN) program. The NCTN gives funds and other support to cancer research organizations to conduct cancer clinical trials.

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Environmental conditions analysis program

International Nuclear Information System (INIS)

Holten, J.

1991-01-01

The PC-based program discussed in this paper has the capability of determining the steady state temperatures of environmental zones (rooms). A program overview will be provided along with examples of formula use. Required input and output from the program will also be discussed. Specific application of plant monitored temperatures and utilization of this program will be offered. The presentation will show how the program can project individual room temperature profiles without continual temperature monitoring of equipment. A discussion will also be provided for the application of the program generated data. Evaluations of anticipated or planned plant modifications and the use of the subject program will also be covered

Sexual Health Education in Massage Therapy Programs: A Survey of Program Directors

Science.gov (United States)

Zamboni, Brian D.; Healey, Dale K.

2016-01-01

Massage therapy program directors completed an online survey to explore sexual education in massage therapy programs. The overall data suggest that program directors are supportive of sexual health education in the training of massage therapists and that such education is integrated into several aspects of their training programs. To enhance…

Vehicle Technologies Program Overview

Energy Technology Data Exchange (ETDEWEB)

none,

2006-09-05

Overview of the Vehicle Technologies Program including external assessment and market view; internal assessment, program history and progress; program justification and federal role; program vision, mission, approach, strategic goals, outputs, and outcomes; and performance goals.

Foreign criteria and programs

International Nuclear Information System (INIS)

Swinth, K.L.; Thompson, I.M.G.

1993-03-01

The concept of measurement quality assurance (MQA) as embodied in National Institute of Standards and Technology (NIST) programs is not generally used within European programs for testing or accreditation. Although the essential elements of quality control and quality assurance are in the European programs, the concept of testing the capability of the laboratory itself, in terms of its performance for the designated measurements, may not be included. Rather, the European programs use the concept of periodic calibration of laboratory reference standards against the next highest level of standards. Thus, they embody the concept of measurement traceability to appropriate primary standards. Within Europe a series of calibration accreditation programs has been established in the various countries tied together through a multilateral agreement. The radiation measurement programs are based on the International Organization for Standardization (ISO) 9000 series of standards. The purpose of this paper is to outline the overall operation of European Accreditation Programs in the radiation calibration and measurement areas. The operation of the radiation measurement programs of the National Measurement Accreditation Service (NAMAS) in the United Kingdom is described in detail along with other European programs. The manner in which these programs relate to individual dosimetry service programs is also described

Developmental Programming of Renal Function and Re-Programming Approaches.

Science.gov (United States)

Nüsken, Eva; Dötsch, Jörg; Weber, Lutz T; Nüsken, Kai-Dietrich

2018-01-01

Chronic kidney disease affects more than 10% of the population. Programming studies have examined the interrelationship between environmental factors in early life and differences in morbidity and mortality between individuals. A number of important principles has been identified, namely permanent structural modifications of organs and cells, long-lasting adjustments of endocrine regulatory circuits, as well as altered gene transcription. Risk factors include intrauterine deficiencies by disturbed placental function or maternal malnutrition, prematurity, intrauterine and postnatal stress, intrauterine and postnatal overnutrition, as well as dietary dysbalances in postnatal life. This mini-review discusses critical developmental periods and long-term sequelae of renal programming in humans and presents studies examining the underlying mechanisms as well as interventional approaches to "re-program" renal susceptibility toward disease. Clinical manifestations of programmed kidney disease include arterial hypertension, proteinuria, aggravation of inflammatory glomerular disease, and loss of kidney function. Nephron number, regulation of the renin-angiotensin-aldosterone system, renal sodium transport, vasomotor and endothelial function, myogenic response, and tubuloglomerular feedback have been identified as being vulnerable to environmental factors. Oxidative stress levels, metabolic pathways, including insulin, leptin, steroids, and arachidonic acid, DNA methylation, and histone configuration may be significantly altered by adverse environmental conditions. Studies on re-programming interventions focused on dietary or anti-oxidative approaches so far. Further studies that broaden our understanding of renal programming mechanisms are needed to ultimately develop preventive strategies. Targeted re-programming interventions in animal models focusing on known mechanisms will contribute to new concepts which finally will have to be translated to human application. Early

Evaluasi Program-program Pengentasan Kemiskinan di Provinsi Bali

Directory of Open Access Journals (Sweden)

AAIN Marhaeni

2014-07-01

Full Text Available The success of the development carried out by a country, including Indonesia, one of which can be seen from the number of poor people. The results of the development carried out by both the local government and the central government still leaving the poverty in most of the existing population. The number of poor people in the Province of Bali, both in absolute and in the percentage is fluctuating, even  absolutely, the number of  poor people in 2011 was much more than in 2009 (183.1 thousand versus 173.6 thousand of people. It is essential to evaluate the government programs to be in accordance with the Act Number 25 Year 2004 on National Development Planning System which mandates the control and evaluation of the implementation of development plans and to be in accordance with the Government Regulation. 39 of 2006, concerning the monitoring. Seeing the importance of activities to perform the evaluation or monitoring, the study aims: 1 to determine the effectiveness of poverty eradication programs that have been implemented by the government so far, which is reducing the expenditure of the poor people, which involving inputs, processes and outputs;2 To analyze the constraints faced by the implementers in the implementation of poverty eradication programs that are reducing the expenditure of the poor; 3 To assess the benefits perceived by the recipients of the program, during receiving aids in the areas of education, health, and food sector; 4 to analyze the weaknesses that exist in the various poverty eradication programs that have been implemented by the government; 5 to assess the commitment of the program implementers in implementing poverty eradication programs in accordance with the community expectations. The research was conducted in three regencies, namely Buleleng, Badung, and Klungkung and each consisted of as many as 90 persons including the recipients of poverty eradication programs in the fields of education, health, and, food

Morning Programs Revisited.

Science.gov (United States)

Black, Susan

1993-01-01

Like British morning programs of recent decades, upstate New York program encourages parents and community residents to get involved in children's education. Parents and community residents--watercolor artists, auto mechanics, doll collectors, and others--are welcomed and valued in schools. Program's purpose is learning, not entertainment. Topics…

Modern programming language

Science.gov (United States)

Feldman, G. H.; Johnson, J. A.

1980-01-01

Structural-programming language is especially-tailored for producing assembly language programs for MODCOMP II and IV mini-computes. Modern programming language consists of set of simple and powerful control structures that include sequencing alternative selection, looping, sub-module linking, comment insertion, statement continuation, and compilation termination capabilities.

Measurement control program

International Nuclear Information System (INIS)

Anon.

1981-01-01

A measurement control program for the model plant is described. The discussion includes the technical basis for such a program, the application of measurement control principles to each measurement, and the use of special experiments to estimate measurement error parameters for difficult-to-measure materials. The discussion also describes the statistical aspects of the program, and the documentation procedures used to record, maintain, and process the basic data. The purpose of the session is to enable participants to: (1) understand the criteria for this type of a measurement control program; (2) understand the kinds of physical standards required for the various measurement processes, e.g., weighing, analytical, NDA; (3) understand the need for and importance of a measurement control program; (4) understand the need for special experiments to provide an improved basis for the measurement of difficult-to-measure materials; (5) understand the general scope of the program's statistical aspects; and (6) understand the basis and scope of the documentation procedures

Gender and Programming.

Science.gov (United States)

Crittenden, Courtney A; Koons-Witt, Barbara A

2017-05-01

The current study examines U.S. prison programming availability and participation by gender on a national level. The authors build upon previous literature by using national-level data, something that has been done in very limited cases previously. The main concern of this study is gender and its effects on programming availability and participation. The U.S. corrections field has undergone major changes in regard to population trends, fiscal constraints, policies, and research over the last few decades without a large-scale examination of the effects of these changes on programming across the United States. In this study, multiple types of programming areas were examined and results indicated that often female prisons (i.e., prisons housing only females) were more likely to offer programs (e.g., mental health options) and women were more likely to participate in many programming options compared with male prisons and men, respectively. We discuss the possible reasons for this and implications for future research.

Evaluation Program initiative

International Nuclear Information System (INIS)

Rich, B.L.

1987-01-01

The purpose of this paper is to provide the Department of Energy's (DOE) safeguards and security community with some insights on an important management initiative by the Office of Security Evaluations (OSE). The paper will present the ''what, where, who, when, and why'' of a new Evaluation Program. The Evaluation Program will be comprised of a continuing series of regular and special evaluations of DOE safeguards and security programs and policies. The evaluations will be integrative and ''crosscutting,'' i.e. will extend across DOE organizational lines. Evaluations will be offered as positive advisories to DOE managers with safeguards and security responsibilities and will not be rated. They will complement the ongoing OSE Inspection Program of inspections conducted by OSE's Inspection Division. The purpose for the evaluations is to establish an accurate and current assessment of the effectiveness and status of safeguards and security programs and policies and to provide DOE managers with required information on program and policy effectiveness

Low-level waste management program: technical program overview

International Nuclear Information System (INIS)

Lowrie, R.S.

1981-01-01

The mission of the technical program is to develop the technology component of the Department of Energy's Low-Level Waste Management Program and to manage research and development, demonstration, and documentation of the technical aspects of the program. Some of the major technology objectives are: develop and demonstrate techniques for waste generation reduction; develop and demonstrate waste treatment, handling and packaging techniques; develop and demonstrate the technology for greater confinement; and develop the technology for remedial action at existing sites. In addition there is the technology transfer objective which is to compile and issue a handbook documenting the technology for each of the above technology objectives

Object-oriented Programming Laws for Annotated Java Programs

Directory of Open Access Journals (Sweden)

Gabriel Falconieri Freitas

2010-03-01

Full Text Available Object-oriented programming laws have been proposed in the context of languages that are not combined with a behavioral interface specification language (BISL. The strong dependence between source-code and interface specifications may cause a number of difficulties when transforming programs. In this paper we introduce a set of programming laws for object-oriented languages like Java combined with the Java Modeling Language (JML. The set of laws deals with object-oriented features taking into account their specifications. Some laws deal only with features of the specification language. These laws constitute a set of small transformations for the development of more elaborate ones like refactorings.

Ostomy Home Skills Program

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Full Text Available ... Trauma Trauma Programs Trauma Programs About Trauma Programs Violence Prevention BleedingControl.org Trauma Quality Programs National Trauma ... Benefits Current Openings Newsroom Newsroom Newsroom Press Releases Media Resources The FIRST Trial ACS Publications ACS in ...

Ostomy Home Skills Program

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Full Text Available ... and Safety Conference Participant Use Data File Surgical Risk Calculator Frequently Asked Questions Participant Hub Contact Us ... Trauma Programs Trauma Programs About Trauma Programs Violence Prevention BleedingControl.org Trauma Quality Programs National Trauma Data ...

Ostomy Home Skills Program

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Program management system manual

International Nuclear Information System (INIS)

1989-08-01

OCRWM has developed a program management system (PMS) to assist in organizing, planning, directing and controlling the Civilian Radioactive Waste Management Program. A well defined management system is necessary because: (1) the Program is a complex technical undertaking with a large number of participants, (2) the disposal and storage facilities to be developed by the Program must be licensed by the Nuclear Regulatory Commission (NRC) and hence are subject to rigorous quality assurance (QA) requirements, (3) the legislation mandating the Program creates a dichotomy between demanding schedules of performance and a requirement for close and continuous consultation and cooperation with external entities, (4) the various elements of the Program must be managed as parts of an integrated waste management system, (5) the Program has an estimated total system life cycle cost of over $30 billion, and (6) the Program has a unique fiduciary responsibility to the owners and generators of the nuclear waste for controlling costs and minimizing the user fees paid into the Nuclear Waste Fund. This PMS Manual is designed and structured to facilitate strong, effective Program management by providing policies and requirements for organizing, planning, directing and controlling the major Program functions

EZLP: An Interactive Computer Program for Solving Linear Programming Problems. Final Report.

Science.gov (United States)

Jarvis, John J.; And Others

Designed for student use in solving linear programming problems, the interactive computer program described (EZLP) permits the student to input the linear programming model in exactly the same manner in which it would be written on paper. This report includes a brief review of the development of EZLP; narrative descriptions of program features,…

Self-patching firmware program

International Nuclear Information System (INIS)

Stanchi, L.

1976-12-01

A method of branching to an auxiliary program without any hardware or software modification at the time the auxiliary program will be written is described. Suitable statements in the main program are already prepared to accept a future program that will be written in machine language. The main program is stored as firmware in PROM memory, while the auxiliary program is loaded into RAM memory. The method is described for the Intel 8080 microprocessor, but can be generalized for any microprocessor or minicomputer that uses ROMs or PROMs. 1 figure, 2 tables

Revealing the programming process

DEFF Research Database (Denmark)

Bennedsen, Jens; Caspersen, Michael Edelgaard

2005-01-01

One of the most important goals of an introductory programming course is that the students learn a systematic approach to the development of computer programs. Revealing the programming process is an important part of this; however, textbooks do not address the issue -- probably because...... the textbook medium is static and therefore ill-suited to expose the process of programming. We have found that process recordings in the form of captured narrated programming sessions are a simple, cheap, and efficient way of providing the revelation.We identify seven different elements of the programming...

Human Reliability Program Overview

Energy Technology Data Exchange (ETDEWEB)

Bodin, Michael

2012-09-25

This presentation covers the high points of the Human Reliability Program, including certification/decertification, critical positions, due process, organizational structure, program components, personnel security, an overview of the US DOE reliability program, retirees and academia, and security program integration.

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Programming Language Pragmatics

CERN Document Server

Scott, Michael L

2009-01-01

Programming Language Pragmatics is the most comprehensive programming language textbook available today. Taking the perspective that language design and language implementation are tightly interconnected, and that neither can be fully understood in isolation, this critically acclaimed and bestselling book has been thoroughly updated to cover the most recent developments in programming language design. With a new chapter on run-time program management and expanded coverage of concurrency, this new edition provides both students and professionals alike with a solid understanding of the most impo

Electron trajectory program

International Nuclear Information System (INIS)

Herrmannsfeldt, W.B.

1979-11-01

The SLAC Electron Trajectory Program is described and instructions and examples for users are given. The program is specifically written to compute trajectories of charged particles in electrostatic and magnetostatic focusing systems including the effects of space charge and self-magnetic fields. Starting options include Child's Law conditions on cathodes of various shapes. Either rectangular or cylindrically symmetric geometry may be used. Magntic fields may be specified using arbitrary configurations of coils, or the output of a magnet program such as Poisson or by an externally calculated array of the axial fields. The program is available in IBM FORTRAN but can be easily converted for use on other brands of hardware. The program is intended to be used with a plotter whose interface the user must provide

Program summary. Nuclear waste management and fuel cycle programs

International Nuclear Information System (INIS)

1982-07-01

This Program Summary Document describes the US Department of Energy (DOE) Nuclear Waste Management and Fuel Cycle Programs. Particular emphasis is given to near-term, specifically Fiscal Year (FY) 1982, activities. The overall objective of these programs will be achieved by the demonstration of: (1) safe radioactive waste management practices for storage and disposal of high-level waste and (2) advanced technologies necessary to close the nuclear fuel cycle on a schedule which would assure a healthy future for the development of nuclear power in this country

FY-2007 PNNL Voluntary Protection Program (VPP) Program Evaluation

International Nuclear Information System (INIS)

Wright, Patrick A.; Fisher, Julie A.; Goheen, Steven C.; Isern, Nancy G.; Madson, Vernon J.; Meicenheimer, Russell L.; Pugh, Ray; Schneirla, Keri A.; Shockey, Loretta L.; Tinker, Mike R.

2008-01-01

This document reports the results of the FY-2007 PNNL VPP Program Evaluation, which is a self-assessment of the operational and programmatic performance of the Laboratory related to worker safety and health. The report was compiled by a team of worker representatives and safety professionals who evaluated the Laboratory's worker safety and health programs on the basis of DOE-VPP criteria. The principle elements of DOE's VPP program are: Management Leadership, Employee Involvement, Worksite Analysis, Hazard Prevention and Control, and Safety and Health Training.

Programming Scala Scalability = Functional Programming + Objects

CERN Document Server

Wampler, Dean

2009-01-01

Learn how to be more productive with Scala, a new multi-paradigm language for the Java Virtual Machine (JVM) that integrates features of both object-oriented and functional programming. With this book, you'll discover why Scala is ideal for highly scalable, component-based applications that support concurrency and distribution. Programming Scala clearly explains the advantages of Scala as a JVM language. You'll learn how to leverage the wealth of Java class libraries to meet the practical needs of enterprise and Internet projects more easily. Packed with code examples, this book provides us

The Development of a Program Engagement Theory for Group Offending Behavior Programs.

Science.gov (United States)

Holdsworth, Emma; Bowen, Erica; Brown, Sarah; Howat, Douglas

2017-10-01

Offender engagement in group offending behavior programs is poorly understood and under-theorized. In addition, there is no research on facilitators' engagement. This article presents the first ever theory to address this gap. A Program Engagement Theory (PET) was derived from a constructivist grounded theory analysis that accounts for both facilitators' and offenders' engagement in group offending behavior programs (GOBPs). Interviews and session observations were used to collect data from 23 program facilitators and 28 offenders (group members). The analysis revealed that group members' engagement involved shared identities and moving on as a group. In turn, this was dependent on facilitators personalising treatment frameworks and establishing a hook to help group members move on. The PET emphasizes the importance of considering change during treatment as a process rather than simply a program outcome. Solution-focused (SF) programs were more conducive to engagement and the change process than offence-focused programs.

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Head First Programming A learner's guide to programming using the Python language

CERN Document Server

Griffiths, David

2009-01-01

Looking for a reliable way to learn how to program on your own, without being overwhelmed by confusing concepts? Head First Programming introduces the core concepts of writing computer programs -- variables, decisions, loops, functions, and objects -- which apply regardless of the programming language. This book offers concrete examples and exercises in the dynamic and versatile Python language to demonstrate and reinforce these concepts. Learn the basic tools to start writing the programs that interest you, and get a better understanding of what software can (and cannot) do. When you're fi

Construction program management

CERN Document Server

Delaney, Joseph

2013-01-01

Although construction is one of the largest industries in the United States, it lags behind other industries in its implementation of modern management techniques such as those contained in the Standard for Program Management (the Standard) by the Project Management Institute (PMI(R)). Construction Program Management details the successful use of the PMI(R) approach for the construction of capital programs. It demonstrates, through case studies, how implementation of PMI's set of tools and techniques can improve the chances of program success. Exploring tactical and strategic management method

Programming language structures

CERN Document Server

Organick, Elliott Irving; Plummer, Robert P

1978-01-01

Programming Language Structures deals with the structures of programming languages and introduces the reader to five important programming languages: Algol, Fortran, Lisp, Snobol, and Pascal. The fundamental similarities and differences among these languages are discussed. A unifying framework is constructed that can be used to study the structure of other languages, such as Cobol, PL/I, and APL. Several of the tools and methodologies needed to construct large programs are also considered.Comprised of 10 chapters, this book begins with a summary of the relevant concepts and principles about al

Customer Service Programming

Science.gov (United States)

1982-01-01

Use of computer program STRCMACS has enabled Illinois Bell Telephone, a subsidiary of American Telephone and Telegraph to cut software development costs about 10 percent by reducing program maintenance and by allowing the department to bring other software into operation more quickly. It has also been useful in company training of programming staff.

Special Milk Program

Science.gov (United States)

US Department of Agriculture, 2009

2009-01-01

The Special Milk Program provides milk to children in schools, child care institutions and eligible camps that do not participate in other Federal child nutrition meal service programs. The program reimburses schools and institutions for the milk they serve. In 2008, 4,676 schools and residential child care institutions participated, along with…

Programming

OpenAIRE

Jackson, M A

1982-01-01

The programmer's task is often taken to be the construction of algorithms, expressed in hierarchical structures of procedures: this view underlies the majority of traditional programming languages, such as Fortran. A different view is appropriate to a wide class of problem, perhaps including some problems in High Energy Physics. The programmer's task is regarded as having three main stages: first, an explicit model is constructed of the reality with which the program is concerned; second, thi...

A Simple Application Program Interface for Saving Java Program Data on a Wiki

OpenAIRE

Yamanoue, Takashi; Oda, Kentaro; Shimozono, Koichi

2012-01-01

A simple application program interface (API) for Java programs running on a wiki is implemented experimentally. A Java program with the API can be running on a wiki, and the Java program can save its data on the wiki. The Java program consists of PukiWiki, which is a popular wiki in Japan, and a plug-in, which starts up Java programs and classes of Java. A Java applet with default access privilege cannot save its data at a local host. We have constructed an API of applets for easy and unified...

The standard for program management

CERN Document Server

2017-01-01

The Standard for Program Management – Fourth Edition differs from prior editions by focusing on the principles of good program management. Program activities have been realigned to program lifecycle phases rather than topics, and the first section was expanded to address the key roles of program manager, program sponsor and program management office. It has also been updated to better align with PMI’s Governance of Portfolios, Programs, and Projects: A Practice Guide.

FY-2007 PNNL Voluntary Protection Program (VPP) Program Evaluation

Energy Technology Data Exchange (ETDEWEB)

Wright, Patrick A.; Fisher, Julie A.; Goheen, Steven C.; Isern, Nancy G.; Madson, Vernon J.; Meicenheimer, Russell L.; Pugh, Ray; Schneirla, Keri A.; Shockey, Loretta L.; Tinker, Mike R.

2008-08-15

This document reports the results of the FY-2007 PNNL VPP Program Evaluation, which is a self-assessment of the operational and programmatic performance of the Laboratory related to worker safety and health. The report was compiled by a team of worker representatives and safety professionals who evaluated the Laboratory's worker safety and health programs on the basis of DOE-VPP criteria. The principle elements of DOE's VPP program are: Management Leadership, Employee Involvement, Worksite Analysis, Hazard Prevention and Control, and Safety and Health Training.

Commercial Midstream Energy Efficiency Incentive Programs: Guidelines for Future Program Design, Implementation, and Evaluation

Energy Technology Data Exchange (ETDEWEB)

Milostan, Catharina [Argonne National Lab. (ANL), Argonne, IL (United States); Levin, Todd [Argonne National Lab. (ANL), Argonne, IL (United States); Muehleisen, Ralph T. [Argonne National Lab. (ANL), Argonne, IL (United States); Guzowski, Leah Bellah B. [Argonne National Lab. (ANL), Argonne, IL (United States)

2017-01-01

Many electric utilities operate energy efficiency incentive programs that encourage increased dissemination and use of energy-efficient (EE) products in their service territories. The programs can be segmented into three broad categories—downstream incentive programs target product end users, midstream programs target product distributors, and upstream programs target product manufacturers. Traditional downstream programs have had difficulty engaging Small Business/Small Portfolio (SBSP) audiences, and an opportunity exists to expand Commercial Midstream Incentive Programs (CMIPs) to reach this market segment instead.

Relationship between internal medicine program board examination pass rates, accreditation standards, and program size.

Science.gov (United States)

Falcone, John L; Gonzalo, Jed D

2014-01-19

To determine Internal Medicine residency program compliance with the Accreditation Council for Graduate Medical Education 80% pass-rate standard and the correlation between residency program size and performance on the American Board of Internal Medicine Certifying Examination. Using a cross-sectional study design from 2010-2012 American Board of Internal Medicine Certifying Examination data of all Internal Medicine residency pro-grams, comparisons were made between program pass rates to the Accreditation Council for Graduate Medical Education pass-rate standard. To assess the correlation between program size and performance, a Spearman's rho was calculated. To evaluate program size and its relationship to the pass-rate standard, receiver operative characteristic curves were calculated. Of 372 Internal Medicine residency programs, 276 programs (74%) achieved a pass rate of =80%, surpassing the Accreditation Council for Graduate Medical Education minimum standard. A weak correlation was found between residency program size and pass rate for the three-year period (p=0.19, pInternal Medicine residency programs complied with Accreditation Council for Graduate Medical Education pass-rate standards, a quarter of the programs failed to meet this requirement. Program size is positively but weakly associated with American Board of Internal Medicine Certifying Examination performance, suggesting other unidentified variables significantly contribute to program performance.

Hemovigilance Program-India

Directory of Open Access Journals (Sweden)

Akanksha Bisht

2013-01-01

Full Text Available A centralized hemovigilance program to assure patient safety and to promote public health has been launched for the first time in India on Dec 10, 2012 in 60 medical colleges in the first phase along with a well-structured program for monitoring adverse reactions associated with blood transfusion and blood product administration. National Institute of Biologicals (NIB will be the National Coordinating Centre for Hemovigilance. This program will be implemented under overall ambit of Pharmacovigilance Program of India (PvPI, which is being coordinated by Indian Pharmacopoeia Commission (IPC. All medical colleges of the country will be enrolled in this program by the year 2016 in order to have a National Centre of Excellence for Hemovigilance at NIB, which will act as a global knowledge platform.

Electron trajectory program

Energy Technology Data Exchange (ETDEWEB)

Herrmannsfeldt, W.B.

1979-11-01

The SLAC Electron Trajectory Program is described and instructions and examples for users are given. The program is specifically written to compute trajectories of charged particles in electrostatic and magnetostatic focusing systems including the effects of space charge and self-magnetic fields. Starting options include Child's Law conditions on cathodes of various shapes. Either rectangular or cylindrically symmetric geometry may be used. Magntic fields may be specified using arbitrary configurations of coils, or the output of a magnet program such as Poisson or by an externally calculated array of the axial fields. The program is available in IBM FORTRAN but can be easily converted for use on other brands of hardware. The program is intended to be used with a plotter whose interface the user must provide.

Rural Electrification Program in Indonesia: Comparing SEHEN and SHS Program

Directory of Open Access Journals (Sweden)

Maxensius Tri Sambodo

2015-07-01

Full Text Available In 2014, the Indonesian government has targets to obtain 80% of electrification ratio and 98.9% of rural electrification ratio. Extending the grid and off-grid connection has been done to obtain the targets. This paper aims to compare two main programs on rural electrification namely Super Extra Energy Saving (Super Ekstra Hemat Energi, SEHEN that is belong to PLN (state owned company in electricity and the Solar Home System (SHS that is financed by the Ministry of Energy and Mineral Resources (MEMR. Indonesia has started the rural electrification program in the late 1950s, but how to provide electricity in a sustainable ways both organizationally and institutionally still become a big challenge. The experiences from East Nusa Tenggara provinces showed that both SEHEN and SHS can instantly improve electrification ratio, but government needs to synchronize the technical, administrative, and financial aspect from the two programs. Without any improvements in designing the program, we argue that the existing program is not sustainable.

HEALS Hypertension Control Program: Training Church Members as Program Leaders.

Science.gov (United States)

Dodani, Sunita; Beayler, Irmatine; Lewis, Jennifer; Sowders, Lindsey A

2014-01-01

Health disparities related to cardiovascular diseases (CVDs) including stroke have remained higher in the African-Americans (AAs) than in other populations. HEALS is a faith-based hypertension (HTN) control program modified according to AA community needs, and delivered by the church-lay members called church health advisors (CHAs). This study examined the feasibility and acceptability of training CHAs as HEALS program leaders. Four CHAs completed a 10-hour HEALS program training workshop at the Church, conducted by the nutrition experts. Workshop was evaluated by CHAs on their level of satisfaction, clarity of contents covered and comfort in delivery the program to the church congregation. The overall six main HEALS curriculum components were completed. Workshop was highly evaluated by CHAs on length of training, balance between content and skills development, and level of satisfaction with program delivery. Church-based culturally modified health promotion interventions conducted by the community lay members may be a way to reduce health disparities in ethnic minorities.

YRE Program Guide.

Science.gov (United States)

California State Dept. of Education, Sacramento. Div. of School Facilities Planning.

This report examines single- and multi-track educational programs as found in California's public school system, explores the pros and cons of using year-round education (YRE) programs, and how to implement these programs. Each year-round tracking system is detailed using each of their calendars in a side-by-side comparison along with the…

Python to learn programming

Science.gov (United States)

Bogdanchikov, A.; Zhaparov, M.; Suliyev, R.

2013-04-01

Today we have a lot of programming languages that can realize our needs, but the most important question is how to teach programming to beginner students. In this paper we suggest using Python for this purpose, because it is a programming language that has neatly organized syntax and powerful tools to solve any task. Moreover it is very close to simple math thinking. Python is chosen as a primary programming language for freshmen in most of leading universities. Writing code in python is easy. In this paper we give some examples of program codes written in Java, C++ and Python language, and we make a comparison between them. Firstly, this paper proposes advantages of Python language in relation to C++ and JAVA. Then it shows the results of a comparison of short program codes written in three different languages, followed by a discussion on how students understand programming. Finally experimental results of students' success in programming courses are shown.

The evolution of programs

CERN Document Server

Dershowitz, Nachum

1983-01-01

-Ecclesiastes 12:12 Programs are invariably subjected to many rorms or transrormation. After an initial version of a program has been designed and developed, it undergoes debugging and certification. In addition, most long-lived pro­ grams have a liCe-cycle that includes modifications to meet amended specifications and extensions for expanded capabilities. Such evolution­ ary aspects of programming are the topic of this monograph. We present rormal methods for manipulating programs and illustrate their applica­ tion with numerous examples. Such methods could be incorporated in semi-automated programming environments, where they would serve to ease the burden on the programmer. We begin by describing a method whereby a given program that achieves one goal can be modified to achieve a different goal or a pro­ gram that computes wrong results can be debugged to achieve the 2 Preface intended results. The abstraction of a set of cognate programs to obtain a program schema, and the instantiation of abstract sc...

Magnetic Fusion Science Fellowship program: Summary of program activities for calendar year 1986

International Nuclear Information System (INIS)

1986-01-01

This report describes the 1985-1986 progress of the Magnetic Fusion Science Fellowship program (MFSF). The program was established in January of 1985 by the Office of Fusion Energy (OFE) of the US Department of Energy (DOE) to encourage talented undergraduate and first-year graduate students to enter qualified graduate programs in the sciences related to fusion energy development. The program currently has twelve fellows in participating programs. Six new fellows are being appointed during each of the program's next two award cycles. Appointments are for one year and are renewable for two additional years with a three year maximum. The stipend level also continues at a $1000 a month or $12,000 a year. The program pays all tuition and fee expenses for the fellows. Another important aspect of the fellowship program is the practicum. During the practicum fellows receive three month appointments to work at DOE designated fusion science research and development centers. The practicum allows the MFSF fellows to directly participate in on-going DOE research and development programs

Programming Python

CERN Document Server

Lutz, Mark

2011-01-01

If you've mastered Python's fundamentals, you're ready to start using it to get real work done. Programming Python will show you how, with in-depth tutorials on the language's primary application domains: system administration, GUIs, and the Web. You'll also explore how Python is used in databases, networking, front-end scripting layers, text processing, and more. This book focuses on commonly used tools and libraries to give you a comprehensive understanding of Python's many roles in practical, real-world programming. You'll learn language syntax and programming techniques in a clear and co

Maintenance procedure upgrade programs

International Nuclear Information System (INIS)

Campbell, J.J.; Zimmerman, C.M.

1988-01-01

This paper describes a systematic approach to upgrading nuclear power plant maintenance procedures. The approach consists of four phases: diagnosis, program planning, program implementation, and program evaluation. Each phase is explained as a series of steps to ensure that all factors in a procedure upgrade program are considered

BeHealthy Charities Aid Foundation Program, Russia: a Program Impact Pathways (PIP) analysis.

Science.gov (United States)

Mukhina, Marina; Novikova, Irina

2014-09-01

In 2007, the Charities Aid Foundation Branch in Russia, under the initiative of and with financial support from the Mondelēz International Foundation and Mondelēz International, launched the charitable BeHealthy Program. The program's main focus is the implementation of four interrelated activities: conducting lessons for schoolchildren on healthy nutrition, with an emphasis on breakfast; healthy cooking lessons with children; cultivating nutritional plants; and providing conditions to encourage children to engage in more physical activity. The program serves more than 13,000 children attending public schools in the Leningrad (Lomonosovskii District), Vladimir, and Novgorod regions. BeHealthy provides funding for schools and comprehensive educational materials to help schoolchildren develop habits of healthy nutrition and physical activity, as well as consulting and expert support for school staff and other key stakeholders. The program brings in experts on program implementation and training for teachers. Curriculum support also includes printed and Web-based healthy lifestyle educational materials on best practices and positive experience, as well as meetings and conferences with school representatives and local authorities. One of the biggest challenges for program managers is to fully understand the complexities of the program, and why and how it is expected to induce changes in healthy lifestyle behaviors of the schoolchildren. For more comprehensive understanding, we performed a Program Impact Pathways (PIP) analysis to identify Critical Quality Control Points (CCPs) and a suite of core indicators of the program's impact on healthy lifestyles. The findings were presented at the Healthy Life-styles Program Evaluation Workshop held in Granada, Spain, 13-14 September 2013, under the auspices of the Mondelēz International Foundation. First, we developed an updated logic model based on how the program was executed. We then translated the logic model into a PIP

Multiobjective programming and planning

CERN Document Server

Cohon, Jared L

2004-01-01

This text takes a broad view of multiobjective programming, emphasizing the methods most useful for continuous problems. It reviews multiobjective programming methods in the context of public decision-making problems, developing each problem within a context that addresses practical aspects of planning issues. Topics include a review of linear programming, the formulation of the general multiobjective programming problem, classification of multiobjective programming methods, techniques for generating noninferior solutions, multiple-decision-making methods, multiobjective analysis of water reso

Introduction to parallel programming

CERN Document Server

Brawer, Steven

1989-01-01

Introduction to Parallel Programming focuses on the techniques, processes, methodologies, and approaches involved in parallel programming. The book first offers information on Fortran, hardware and operating system models, and processes, shared memory, and simple parallel programs. Discussions focus on processes and processors, joining processes, shared memory, time-sharing with multiple processors, hardware, loops, passing arguments in function/subroutine calls, program structure, and arithmetic expressions. The text then elaborates on basic parallel programming techniques, barriers and race

Technology Commercialization Program 1991

Energy Technology Data Exchange (ETDEWEB)

1991-11-01

This reference compilation describes the Technology Commercialization Program of the Department of Energy, Defense Programs. The compilation consists of two sections. Section 1, Plans and Procedures, describes the plans and procedures of the Defense Programs Technology Commercialization Program. The second section, Legislation and Policy, identifies legislation and policy related to the Program. The procedures for implementing statutory and regulatory requirements are evolving with time. This document will be periodically updated to reflect changes and new material.

Successful Bullying Prevention Programs: Influence of Research Design, Implementation Features, and Program Components

Directory of Open Access Journals (Sweden)

Bryanna Hahn Fox

2012-12-01

Full Text Available Bullying prevention programs have been shown to be generally effective in reducing bullying and victimization. However, the effects are relatively small in randomized experiments and greater in quasi-experimental and age-cohort designs. Programs that are more intensive and of longer duration (for both children and teachers are more effective, as are programs containing more components. Several program components are associated with large effect sizes, including parent training or meetings and teacher training. These results should inform the design and evaluation of anti-bullying programs in the future, and a system ofaccreditation of effective programs.

3-D diffusion program XYZ-MUGDI with data-analyzing program XYZFF

Energy Technology Data Exchange (ETDEWEB)

Siewers, H

1976-01-01

The XYZ-MUGDI program solves boudary value and eigenvalue problems for three-dimensional heterogeneous configurations in X, Y, Z-geometry for a maximum of four energy groups. The program XYZFF is a program for analyzing data; with it especially the average and maximum form factors for arbitrary coarse-meshed networks can be evaluated.

A Review of Generic Program Visualization Systems for Introductory Programming Education

Science.gov (United States)

Sorva, Juha; Karavirta, Ville; Malmi, Lauri

2013-01-01

This article is a survey of program visualization systems intended for teaching beginners about the runtime behavior of computer programs. Our focus is on generic systems that are capable of illustrating many kinds of programs and behaviors. We inclusively describe such systems from the last three decades and review findings from their empirical…

Knowledge management program

International Nuclear Information System (INIS)

Henderson, T.

2013-01-01

To capture and retain the CANDU experience from a wider CANDU base and transfer knowledge and experience to our members, supplier participants and universities in a cost effective manner. Major focus area of the program is knowledge management joint projects, generic training delivery, inter-utilities mentoring and technical support, public education programs. The path forward is execution of transition of OPG NPDS Program as an ongoing program in COG with member funding, pursue opportunities to provide member utilities with additional leadership and train-the-trainer training and grow the knowledge management activities by 20% per year based on 2013/2014 results.

Practical C programming

CERN Document Server

Oualline, Steve

1997-01-01

There are lots of introductory C books, but this is the first one that has the no-nonsense, practical approach that has made Nutshell Handbooks® famous. C programming is more than just getting the syntax right. Style and debugging also play a tremendous part in creating programs that run well and are easy to maintain. This book teaches you not only the mechanics of programming, but also describes how to create programs that are easy to read, debug, and update. Practical rules are stressed. For example, there are fifteen precedence rules in C (&& comes before || comes before ?:). The practi

Knowledge management program

Energy Technology Data Exchange (ETDEWEB)

Henderson, T. [CANDU Owners' Group, Toronto, Ontario (Canada)

2013-07-01

To capture and retain the CANDU experience from a wider CANDU base and transfer knowledge and experience to our members, supplier participants and universities in a cost effective manner. Major focus area of the program is knowledge management joint projects, generic training delivery, inter-utilities mentoring and technical support, public education programs. The path forward is execution of transition of OPG NPDS Program as an ongoing program in COG with member funding, pursue opportunities to provide member utilities with additional leadership and train-the-trainer training and grow the knowledge management activities by 20% per year based on 2013/2014 results.

Canadian fusion program

International Nuclear Information System (INIS)

Brown, T.S.

1982-06-01

The National Research Council of Canada is establishing a coordinated national program of fusion research and development that is planned to grow to a total annual operating level of about $20 million in 1985. The long-term objective of the program is to put Canadian industry in a position to manufacture sub-systems and components of fusion power reactors. In the near term the program is designed to establish a minimum base of scientific and technical expertise sufficient to make recognized contributions and thereby gain access to the international effort. The Canadian program must be narrowly focussed on a few specializations where Canada has special indigenous skills or technologies. The programs being funded are the Tokamak de Varennes, the Fusion Fuels Technology Project centered on tritium management, and high-power gas laser technology and associated diagnostic instrumentation

Refrigerator retirement and replacement programs : lessons learned and application to an Ontario wide program

Energy Technology Data Exchange (ETDEWEB)

NONE

2006-05-15

The best practices in refrigerator retirement programs in North America were identified in an effort to develop a concept for an Ontario-wide provincial refrigerator retirement program. The report focused on describing refrigerator retirement programs, namely those programs that focused on getting rid of old secondary refrigerators. The report excluded refrigerator replacement programs, which encourage householders to retire their refrigerators early and replace them with an energy star refrigerator. However, it was noted that in several regions, both replacement and retirement programs are offered at the same time. The report provided background information on energy use by refrigerators as well as refrigerator retirement and replacement programs. Types of refrigerator retirement and replacement programs and the environmental benefits of these programs were also described. The report also addressed the potential energy impact of an Ontario-wide refrigerator retirement program as well as consumer incentive and bounties initiatives to encourage households to retire units. Other topics covered in the report included the design of typical refrigerator retirement and replacement programs; collection and recycling of retired refrigerators; reported costs of refrigerator retirement and replacement programs; as well as marketing and advertising. The role of retailers and manufacturers and reported lessons learned from refrigerator retirement and replacement were also presented. 14 refs., 6 tabs., 6 appendices.

Program Director Participation in a Leadership and Management Skills Fellowship and Characteristics of Program Quality.

Science.gov (United States)

Carek, Peter J; Mims, Lisa D; Conry, Colleen M; Maxwell, Lisa; Greenwood, Vicki; Pugno, Perry A

2015-01-01

The association between a residency program director completing a leadership and management skills fellowship and characteristics of quality and innovation of his/her residency program has not been studied. Therefore, the aim of this study is to examine the association between a residency program director's completion of a specific fellowship addressing these skills (National Institute for Program Director Development or NIPDD) and characteristics of quality and innovation of the program they direct. Using information from the American Academy of Family Physicians (AAFP), National Resident Matching Program (NRMP) and FREIDA® program characteristics were obtained. Descriptive statistics were used to summarize the data. The relationship between programs with a NIPDD graduate as director and program quality measures and indicators of innovation was analyzed using both chi square and logistic regression. Initial analyses showed significant associations between the NIPDD graduate status of a program director and regional location, mean years of program director tenure, and the program's 5-year aggregate ABFM board pass rate from 2007--2011. After grouping the programs into tertiles, the regression model showed significant positive associations with programs offering international experiences and being a NIPDD graduate. Program director participation in a fellowship addressing leadership and management skills (ie, NIPDD) was found to be associated with higher pass rates of new graduates on a Board certification examination and predictive of programs being in the upper tertile of programs in terms of Board pass rates.

The Program Administrator Cost of Saved Energy for Utility Customer-Funded Energy Efficiency Programs

Energy Technology Data Exchange (ETDEWEB)

Billingsley, Megan A.; Hoffman, Ian M.; Stuart, Elizabeth; Schiller, Steven R.; Goldman, Charles A.; LaCommare, Kristina

2014-03-19

End-use energy efficiency is increasingly being relied upon as a resource for meeting electricity and natural gas utility system needs within the United States. There is a direct connection between the maturation of energy efficiency as a resource and the need for consistent, high-quality data and reporting of efficiency program costs and impacts. To support this effort, LBNL initiated the Cost of Saved Energy Project (CSE Project) and created a Demand-Side Management (DSM) Program Impacts Database to provide a resource for policy makers, regulators, and the efficiency industry as a whole. This study is the first technical report of the LBNL CSE Project and provides an overview of the project scope, approach, and initial findings, including: • Providing a proof of concept that the program-level cost and savings data can be collected, organized, and analyzed in a systematic fashion; • Presenting initial program, sector, and portfolio level results for the program administrator CSE for a recent time period (2009-2011); and • Encouraging state and regional entities to establish common reporting definitions and formats that would make the collection and comparison of CSE data more reliable. The LBNL DSM Program Impacts Database includes the program results reported to state regulators by more than 100 program administrators in 31 states, primarily for the years 2009–2011. In total, we have compiled cost and energy savings data on more than 1,700 programs over one or more program-years for a total of more than 4,000 program-years’ worth of data, providing a rich dataset for analyses. We use the information to report costs-per-unit of electricity and natural gas savings for utility customer-funded, end-use energy efficiency programs. The program administrator CSE values are presented at national, state, and regional levels by market sector (e.g., commercial, industrial, residential) and by program type (e.g., residential whole home programs, commercial new

77 FR 14465 - Public Transportation on Indian Reservations Program; Tribal Transit Program

Science.gov (United States)

2012-03-09

... DEPARTMENT OF TRANSPORTATION Federal Transit Administration Public Transportation on Indian... in funding provided by the Public Transportation on Indian Reservations Program (Tribal Transit... establishing the Public Transportation on Indian Reservations Program (Tribal Transit Program or TTP). This...

Croatian non-fossil energy programme (NFE Program); Program nefosilnih energenata u Hrvatskoj (Program NFE)

Energy Technology Data Exchange (ETDEWEB)

Kamenski, M; Potocnik, V [Zagreb (Croatia)

1997-12-31

Proposed NFE Program (rational energy use and renewable energy) takes into account European Union experience and the facts of scarce fossil fuel reserves and relatively low energy efficiency in Croatia. Implementation of the NFE Program is expected to reduce fossil fuels import growth and environmental pollution, to improve import-export trade balance and to contribute to the new local jobs. (author). 4 figs., 11 refs.

BASIC Programming.

Science.gov (United States)

Jennings, Carol Ann

Designed for use by both secondary- and postsecondary-level business teachers, this curriculum guide consists of 10 units of instructional materials dealing with Beginners All-Purpose Symbol Instruction Code (BASIC) programing. Topics of the individual lessons are numbering BASIC programs and using the PRINT, END, and REM statements; system…

Nutrition Program Quality Assurance through a Formalized Process of On-Site Program Review

Science.gov (United States)

Paddock, Joan Doyle; Dollahite, Jamie

2012-01-01

A protocol for a systematic onsite review of the Expanded Food and Nutrition Education Program and Supplemental Nutrition Assistance Program-Education was developed to support quality programming and ensure compliance with state guidelines and federal regulations. Onsite review of local nutrition program operations is one strategy to meet this…

Which Introductory Programming Approach Is Most Suitable for Students: Procedural or Visual Programming?

Science.gov (United States)

Eid, Chaker; Millham, Richard

2012-01-01

In this paper, we discuss the visual programming approach to teaching introductory programming courses and then compare this approach with that of procedural programming. The involved cognitive levels of students, as beginning students are introduced to different types of programming concepts, are correlated to the learning processes of…

The Value in Evaluating and Communicating Program Impact: The Ohio BR&E Program

Science.gov (United States)

Daivs, Gregory

2012-01-01

Assessing program impact can provide useful program evaluation data. It also provides a basis for program development, marketing, and justification. This article discusses recent impact evaluation efforts and findings of a long-time Extension program; referred to as Business Retention and Expansion (BR&E). How such information can be…

The Trailblazer Program

Science.gov (United States)

Trefney, Charles J.

1999-01-01

This paper presents the "Three Pillars of Success" for the Trailblazer Program. The topics include: 1) The "Rocket Equation" for SSTO (Single Stage To Orbit); 2) The Rocket I* Barrier; 3) Rocket-Based Combined-Cycle Engine; 4) Potential for Reusability; 5) Factors Mitigating RBCC Performance; 6) The "Trailblazer" Program; 7) Trailblazer Performance Goals; 8) Trailblazer Reference Vehicle; and 9) Trailblazer Program Architecture.

Resource programs: Draft Environmental Impact Statement Resource Programs

International Nuclear Information System (INIS)

1992-03-01

Every two years, Bonneville Power Administration (BPA) prepares a Resource Program which identifies the resource actions BPA will take to meet its obligation to serve the forecasted power requirements of its customers. The Resource Program's Environmental Impact Statement (RPEIS) is a programmatic environmental document which will support decisions made in several future Resource Programs. Environmental documents tiered to the EIS may be prepared on a site-specific basis. The RPEIS includes a description of the environmental effects and mitigation for the various resource types available in order to evaluate the trade-offs among them. It also assesses the environmental impacts of adding thirteen alternative combinations of resources to the existing power system. This report contains the appendices to the RPEIS

The Cybernetic Writing Program.

Science.gov (United States)

Lowe, Kelly Fisher

This paper looks at the role of a Writing Program Administrator, and applies the idea of a cybernetic system to the administration of the program. In this cybernetic model, the Writing Program Administrator (WPA) works as both a problem solver and problem causer, with the responsibility of keeping the program in proper balance. A cybernetic…

76 FR 44394 - Public Transportation on Indian Reservations Program; Tribal Transit Program

Science.gov (United States)

2011-07-25

... DEPARTMENT OF TRANSPORTATION Federal Transit Administration Public Transportation on Indian... the Public Transportation on Indian Reservations Program (Tribal Transit Program) (TTP). This program... of the Census and may be used for public transportation capital projects, operating costs of...

Impact of Aspect-Oriented Programming on the Quality of Novices’ Programs: A Comparative Study

Directory of Open Access Journals (Sweden)

Marija Katic

2013-06-01

Full Text Available Aspect-oriented programming has been introduced in order to increase the modularity of object-oriented programs and is claimed to improve software quality. Although there are various researches on this claim, the question to what extent aspect-oriented programming improves the quality of programs depending on a developer’s experience still remains. The purpose of this study is to investigate whether aspect-oriented programming used by novice programmers improves the quality of programs, in terms of software flexibility and readability (consequently reusability and maintainability as well. As a part of an undergraduate course in programming paradigms and languages, a systematic comparison between students’ object-oriented and aspect-oriented solutions of the same problem was driven. In order to drive this comparison we have established the basis for the development of the new quality assessment model consisting of software metrics for an objective evaluation and student survey for subjective evaluation. The results show that the use of aspect-oriented programming lead to novices’ programs that are easier to change and read (flexible and readable compared to object-oriented programs. What is more, administered survey showed that students perceive their programs as more flexible and readable.

Recruiting Fathers to Parenting Programs: Advice from Dads and Fatherhood Program Providers

Science.gov (United States)

Stahlschmidt, Mary Jo; Threlfall, Jennifer; Seay, Kristen D.; Lewis, Ericka M.; Kohl, Patricia L.

2014-01-01

The benefits of high-quality father-child relationships for fathers and children alike are well documented. While evidence suggests parenting programs can improve the quality of father-child relationships, few fathers participate in such programs. This qualitative study aims to fill the gap in knowledge on best practices for recruiting urban African American fathers, a group of fathers with unique parenting challenges, to parenting programs. Focus groups were conducted with 29 fathers to gain their perspectives on recruitment strategies. Semi-structured interviews were also conducted with a nationwide sample of 19 fatherhood program providers to learn about their most successful recruitment strategies. Recruitment strategies based on emergent themes from the focus groups and interviews are presented here. Themes included using word-of-mouth recruitment, increasing advertising, targeting advertising specifically to urban African American fathers, providing transportation and incentives, recruiting through the courts, collaborating with other community agencies, and offering parenting programming along with other programming valued by fathers such as employment assistance. Implications for developing strategies for recruiting urban African American fathers to parenting programs are discussed. PMID:24791035

Recruiting Fathers to Parenting Programs: Advice from Dads and Fatherhood Program Providers.

Science.gov (United States)

Stahlschmidt, Mary Jo; Threlfall, Jennifer; Seay, Kristen D; Lewis, Ericka M; Kohl, Patricia L

2013-10-01

The benefits of high-quality father-child relationships for fathers and children alike are well documented. While evidence suggests parenting programs can improve the quality of father-child relationships, few fathers participate in such programs. This qualitative study aims to fill the gap in knowledge on best practices for recruiting urban African American fathers, a group of fathers with unique parenting challenges, to parenting programs. Focus groups were conducted with 29 fathers to gain their perspectives on recruitment strategies. Semi-structured interviews were also conducted with a nationwide sample of 19 fatherhood program providers to learn about their most successful recruitment strategies. Recruitment strategies based on emergent themes from the focus groups and interviews are presented here. Themes included using word-of-mouth recruitment, increasing advertising, targeting advertising specifically to urban African American fathers, providing transportation and incentives, recruiting through the courts, collaborating with other community agencies, and offering parenting programming along with other programming valued by fathers such as employment assistance. Implications for developing strategies for recruiting urban African American fathers to parenting programs are discussed.

Older Workers and Federal Work Programs: The Korean Senior Employment Program (KSEP).

Science.gov (United States)

Choi, Eunhee

2016-01-01

Federal older worker programs are attracting attention due to the growing number of older workers across the world. They are uniquely situated to provide out-of-market work opportunities to older job seekers, who often find their age a barrier to securing desirable jobs. In 2004, the Korean government established its own program, the Korean Senior Employment Program (KSEP); however, literature for international readers on this innovative program is lacking. Thus, this article aims to provide an in-depth description of KSEP and a brief comparison between the Senior Community Service Employment Program in the U.S. and KSEP. The unique characteristics of KSEP include having the dual program foci on supplemental income and social participation; expanding work opportunities in the private sector beyond community-based jobs; accepting participants who are financially disadvantaged as well as those with a high desire for social participation regardless of their income; and broadening work opportunities for those with professional skills beyond repetitive, simple, and temporary jobs. This article may offer helpful insights to older worker advocates from various countries in creating or modifying their programs.

Summary of Program Evaluation Results: 1985-1986 School Year Pre-Kindergarten Educational Program.

Science.gov (United States)

Heath, Robert W.; And Others

Reported are findings of the 1985-86 program evaluation of the prenatal-to-preschool and preschool programs operating under the auspices of the Kamehameha Schools/Bishop Estate. Evaluation of the prenatal-to-preschool program (the Kupulani Program) included item analysis of the Questions about Pregnancy Test, development of a revised data…

Exploring the Educational Benefits of Introducing Aspect-Oriented Programming Into a Programming Course

Science.gov (United States)

Boticki, I.; Katic, M.; Martin,S.

2013-01-01

This paper explores the educational benefits of introducing the aspect-oriented programming paradigm into a programming course in a study on a sample of 75 undergraduate software engineering students. It discusses how using the aspect-oriented paradigm, in addition to the object-oriented programming paradigm, affects students' programs, their exam…

A protein-based set of reference markers for liver tissues and hepatocellular carcinoma

International Nuclear Information System (INIS)

Sun, Stella; Yi, Xin; Poon, Ronnie TP; Yeung, Chun; Day, Philip JR; Luk, John M

2009-01-01

During the last decade, investigations have focused on revealing genes or proteins that are involved in HCC carcinogenesis using either genetic or proteomic techniques. However, these studies are overshadowed by a lack of good internal reference standards. The need to identify 'housekeeping' markers, whose expression is stable in various experimental and clinical conditions, is therefore of the utmost clinical relevance in quantitative studies. This is the first study employed 2-DE analysis to screen for potential reference markers and aims to correlate the abundance of these proteins with their level of transcript expression. A Chinese cohort of 224 liver tissues samples (105 cancerous, 103 non-tumourous cirrhotic, and 16 normal) was profiled using 2-DE analysis. Expression of the potential reference markers was confirmed by western blot, immunohistochemistry and real-time quantitative PCR. geNorm algorithm was employed for gene stability measure of the identified reference markers. The expression levels of three protein markers beta-actin (ACTB), heat shock protein 60 (HSP60), and protein disulphide isomerase (PDI) were found to be stable using p-values (p > 0.99) as a ranking tool in all 224 human liver tissues examined by 2-DE analysis. Of high importance, ACTB and HSP 60 were successfully validated at both protein and mRNA levels in human hepatic tissues by western blot, immunohistochemistry and real-time quantitative PCR. In addition, no significant correlation of these markers with any clinicopathological features of HCC and cirrhosis was found. Gene stability measure of these two markers with other conventionally applied housekeeping genes was assessed by the geNorm algorithm, which ranked ACTB and HSP60 as the most stable genes among this cohort of clinical samples. Our findings identified 2 reference markers that exhibited stable expression across human liver tissues with different conditions thus should be regarded as reliable reference

Mammography accreditation program

Energy Technology Data Exchange (ETDEWEB)

Wilcox, P.

1993-12-31

In the mid-1980`s, the movement toward the use of dedicated mammography equipment provided significant improvement in breast cancer detection. However, several studies demonstrated that this change was not sufficient to ensure optimal image quality at a low radiation dose. In particular, the 1985 Nationwide Evaluation of X-ray Trends identified the wide variations in image quality and radiation dose, even from dedicated units. During this time period, the American Cancer Society (ACS) launched its Breast Cancer Awareness Screening Campaign. However, there were concerns about the ability of radiology to respond to the increased demand for optimal screening examinations that would result from the ACS program. To respond to these concerns, the ACS and the American College of Radiology (ACR) established a joint committee on mammography screening in 1986. After much discussion, it was decided to use the ACR Diagnostic Practice Accreditation Program as a model for the development of a mammography accreditation program. However, some constraints were required in order to make the program meet the needs of the ACS. This voluntary, peer review program had to be timely and cost effective. It was determined that the best way to address these needs would be to conduct the program by mail. Finally, by placing emphasis on the educational nature of the program, it would provide an even greater opportunity for improving mammographic quality. The result of this effort was that, almost six years ago, in May 1987, the pilot study for the ACR Mammography Accreditation Program (MAP) began, and in August of that year, the first applications were received. In November 1987, the first 3-year accreditation certificates were awarded.

Mammography accreditation program

International Nuclear Information System (INIS)

Wilcox, P.

1993-01-01

In the mid-1980's, the movement toward the use of dedicated mammography equipment provided significant improvement in breast cancer detection. However, several studies demonstrated that this change was not sufficient to ensure optimal image quality at a low radiation dose. In particular, the 1985 Nationwide Evaluation of X-ray Trends identified the wide variations in image quality and radiation dose, even from dedicated units. During this time period, the American Cancer Society (ACS) launched its Breast Cancer Awareness Screening Campaign. However, there were concerns about the ability of radiology to respond to the increased demand for optimal screening examinations that would result from the ACS program. To respond to these concerns, the ACS and the American College of Radiology (ACR) established a joint committee on mammography screening in 1986. After much discussion, it was decided to use the ACR Diagnostic Practice Accreditation Program as a model for the development of a mammography accreditation program. However, some constraints were required in order to make the program meet the needs of the ACS. This voluntary, peer review program had to be timely and cost effective. It was determined that the best way to address these needs would be to conduct the program by mail. Finally, by placing emphasis on the educational nature of the program, it would provide an even greater opportunity for improving mammographic quality. The result of this effort was that, almost six years ago, in May 1987, the pilot study for the ACR Mammography Accreditation Program (MAP) began, and in August of that year, the first applications were received. In November 1987, the first 3-year accreditation certificates were awarded

Abandoned works program

International Nuclear Information System (INIS)

Arnott, A.

2007-01-01

Thousands of improperly abandoned or decommissioned oil and gas wells are threatening the purity of Ontario's source water. This presentation discussed an abandoned works program developed by the Ontario Ministry of Natural Resources. The abandoned works program was established in 2005 in order to plug old oil and gas wells. The program was designed to create a list of abandoned wells, develop a coherent policy, and formulate procurement and contracting protocols. Abandoned wells are defined as wells drilled prior to 1963 with no operator other than the current landowner. There are currently over 200 prioritized wells on the list. Twenty-six contracts have been issued for a total of 33 wells, and 19 wells have been plugged since the program's field operations began in 2006. However, the program is often challenged by the difficulties associated with determining where the wells are located. Many of the wells have been cut off and buried, and access is often dependent on weather conditions and road restrictions. There is also a shortage of contractors who have experience working with older wells. It was concluded that the program will expand by obtaining further funding and modifying its qualification criteria. tabs., figs

Functional Programming in R

DEFF Research Database (Denmark)

Mailund, Thomas

Master functions and discover how to write functional programs in R. In this book, you'll make your functions pure by avoiding side-effects; you’ll write functions that manipulate other functions, and you’ll construct complex functions using simpler functions as building blocks. In Functional...... Programming in R, you’ll see how we can replace loops, which can have side-effects, with recursive functions that can more easily avoid them. In addition, the book covers why you shouldn't use recursion when loops are more efficient and how you can get the best of both worlds. Functional programming...... is a style of programming, like object-oriented programming, but one that focuses on data transformations and calculations rather than objects and state. Where in object-oriented programming you model your programs by describing which states an object can be in and how methods will reveal or modify...

Functional Programming in R

DEFF Research Database (Denmark)

Mailund, Thomas

2017-01-01

Master functions and discover how to write functional programs in R. In this book, you'll make your functions pure by avoiding side-effects; you’ll write functions that manipulate other functions, and you’ll construct complex functions using simpler functions as building blocks. In Functional...... Programming in R, you’ll see how we can replace loops, which can have side-effects, with recursive functions that can more easily avoid them. In addition, the book covers why you shouldn't use recursion when loops are more efficient and how you can get the best of both worlds. Functional programming...... is a style of programming, like object-oriented programming, but one that focuses on data transformations and calculations rather than objects and state. Where in object-oriented programming you model your programs by describing which states an object can be in and how methods will reveal or modify...

CROSSER - CUMULATIVE BINOMIAL PROGRAMS

Science.gov (United States)

Bowerman, P. N.

1994-01-01

The cumulative binomial program, CROSSER, is one of a set of three programs which calculate cumulative binomial probability distributions for arbitrary inputs. The three programs, CROSSER, CUMBIN (NPO-17555), and NEWTONP (NPO-17556), can be used independently of one another. CROSSER can be used by statisticians and users of statistical procedures, test planners, designers, and numerical analysts. The program has been used for reliability/availability calculations. CROSSER calculates the point at which the reliability of a k-out-of-n system equals the common reliability of the n components. It is designed to work well with all integer values 0 < k <= n. To run the program, the user simply runs the executable version and inputs the information requested by the program. The program is not designed to weed out incorrect inputs, so the user must take care to make sure the inputs are correct. Once all input has been entered, the program calculates and lists the result. It also lists the number of iterations of Newton's method required to calculate the answer within the given error. The CROSSER program is written in C. It was developed on an IBM AT with a numeric co-processor using Microsoft C 5.0. Because the source code is written using standard C structures and functions, it should compile correctly with most C compilers. The program format is interactive. It has been implemented under DOS 3.2 and has a memory requirement of 26K. CROSSER was developed in 1988.

76 FR 34541 - Child and Adult Care Food Program Improving Management and Program Integrity

Science.gov (United States)

2011-06-13

... 7 CFR Parts 210, 215, 220 et al. Child and Adult Care Food Program Improving Management and Program..., 220, 225, and 226 RIN 0584-AC24 Child and Adult Care Food Program Improving Management and Program... management and integrity in the Child and Adult Care Food Program (CACFP), at 67 FR 43447 (June 27, 2002) and...

Intelligence of programs

Energy Technology Data Exchange (ETDEWEB)

Novak, D

1982-01-01

A general discussion about the level of artificial intelligence in computer programs is presented. The suitability of various languages for the development of complex, intelligent programs is discussed, considering fourth-generation language as well as the well established structured COBOL language. It is concluded that the success of automation in many administrative fields depends to a large extent on the development of intelligent programs.

Surplus Facilities Management Program

International Nuclear Information System (INIS)

Coobs, J.H.

1983-01-01

This is the second of two programs that are concerned with the management of surplus facilities. The facilities in this program are those related to commercial activities, which include the three surplus experimental and test reactors [(MSRE, HRE-2, and the Low Intensity Test Reactor (LITR)] and seven experimental loops at the ORR. The program is an integral part of the Surplus Facilities Management Program, which is a national program administered for DOE by the Richland Operations Office. Very briefly reported here are routine surveillance and maintenance of surplus radioactively contaminated DOE facilities awaiting decommissioning

Energy research program 82

International Nuclear Information System (INIS)

1982-01-01

The energy research program 82 (EFP-82) is prepared by the Danish ministry of energy in order to continue the extension of the Danish energy research and development started through the former trade ministry's programs EM-1 (1976) and EM-2 (1978), and the energy ministry's programs EFP-80 and EFP-81. The new program is a continuation of the activities in the period 1982-84 with a total budget of 100 mio.Dkr. The program gives a brief description of background, principles, organization and financing, and a detailed description of each research area. (BP)

Computer Programs.

Science.gov (United States)

Anderson, Tiffoni

This module provides information on development and use of a Material Safety Data Sheet (MSDS) software program that seeks to link literacy skills education, safety training, and human-centered design. Section 1 discusses the development of the software program that helps workers understand the MSDSs that accompany the chemicals with which they…

Ostomy Home Skills Program

Medline Plus

Full Text Available ... ACS Careers at ACS About ACS Career Types Working at ACS ... Education Patients and Family Skills Programs Ostomy Home Skills Program Ostomy Home Skills Program Adult Ostomy ...

Programs as Data Objects

DEFF Research Database (Denmark)

This book constitutes the refereed proceedings of the Second Symposium on Programs as Data Objects, PADO 2001, held in Aarhus, Denmark, in May 2001. The 14 revised full papers presented were carefully reviewed and selected from 30 submissions. Various aspects of looking at programs as data objects...... are covered from the point of view of program analysis, program transformation, computational complexity, etc....

MIDI Programming in Scheme

DEFF Research Database (Denmark)

Nørmark, Kurt

2010-01-01

makes it possible to carry out systematic modifications and transformations of MIDI contents with use of pure functional programming. Side by side with the XML-inspired MIDI language, the paper describes an Emacs-based, textual programming environment that supports the MIDI programming process....... The programming environment also supports a variety of interactive features - similar to MIDI sequencers - but restricted to a textual representation of the music. The main contributions of the work are considered to be (1) An accumulated MIDI function library, which can transform MIDI files in many non......-trivial ways; (2) A proposed working process alternating between creative mode and programmatic editing mode within a MIDI programming environment; and (3) A textual MIDI programming environment with embedded support of many interactive, MIDI-related functionalities....

Mondelēz Hope Kitchen Program, China: a Program Impact Pathways (PIP) analysis.

Science.gov (United States)

Li, Yanran; Yao, Xiaoxun; Gu, Lan

2014-09-01

Mondelēz Hope Kitchen is a community program initiated jointly in 2009 by Mondelēz International and the China Youth Development Foundation (CYDF). In response to the urgent needs of students, parents, and teachers at primary and middle schools in poverty-stricken rural areas of China, the program addresses the complex and intertwined issues of undernutrition and obesity. By funding both kitchen equipment and teacher training in health and nutrition, the Mondelēz Hope Kitchen Program improves the capacity of schools to supply healthy meals, helping students to access safe and nutritious foods and, ultimately, to improve their nutritional status and health. In 2011, the Mondelēz International Foundation awarded CYDF a grant to formally assess the impact of the original program design. The Mondelēz International Foundation encouraged CYDF and six other healthy lifestyles-focused community partners around the world to participate in this program evaluation workshop. The goals of this study were to describe the logic model of the Mondelēz Hope Kitchen Program, summarize a recent evaluation of the Mondelēz Hope Kitchen Program, and conduct a Program Impact Pathways (PIP) analysis to identify Critical Quality Control Points (CCPs) and a suite of impact indicators. The findings were presented at the Healthy Lifestyles Program Evaluation Workshop held in Granada, Spain, 13-14 September 2013, under the auspices of the Mondelēz International Foundation. The authors developed the program's PIP diagram based on deliberations involving the program managers and Director and consulting the "Hope Kitchen Management Rules "and "Hope Kitchen Inspection and Acceptance Report". The PIP analyses identified three CCPs: buy-in from schools, kitchen infrastructure, and changes in teachers' knowledge of nutrition after training. In addition, changes in children's knowledge of nutrition will be added to the core suite of impact evaluation indicators that also includes children

A review of pediatric dentistry program websites: what are applicants learning about our programs?

Science.gov (United States)

Lin, Jenn-Yih; Lee, Jung; Davidson, Bo; Farquharson, Kara; Shaul, Cheryl; Kim, Sara

2010-06-01

The purpose of this study was twofold: 1) to examine website content provided by U.S. and Canadian pediatric dentistry residency programs, and 2) to understand aspects of program websites that dental students report to be related to their interests. Sixty-eight program websites were reviewed by five interprofessional evaluators. A thirty-six-item evaluation form was organized into 1) program descriptive items listed on the American Academy of Pediatric Dentistry (AAPD) website (n=21); 2) additional program descriptive items not listed on the AAPD website but of interest (n=9); and 3) items related to website interface design (n=5). We also surveyed fifty-four dental students regarding their interest in various aspects of program descriptions. The results of this study suggest that pediatric dentistry residency programs in general tend to provide identical or less information than what is listed on the AAPD website. The majority of respondents (76 percent) reported that residency program websites would be their first source of information about advanced programs. The greatest gap between the available website information and students' interests exists in these areas: stipend and tuition information, state licensure, and program strengths. Pediatric dentistry residency programs underutilize websites as a marketing and recruitment tool and should incorporate more information in areas of students' priority interests.

Developmental Programming of Renal Function and Re-Programming Approaches

Science.gov (United States)

Nüsken, Eva; Dötsch, Jörg; Weber, Lutz T.; Nüsken, Kai-Dietrich

2018-01-01

Chronic kidney disease affects more than 10% of the population. Programming studies have examined the interrelationship between environmental factors in early life and differences in morbidity and mortality between individuals. A number of important principles has been identified, namely permanent structural modifications of organs and cells, long-lasting adjustments of endocrine regulatory circuits, as well as altered gene transcription. Risk factors include intrauterine deficiencies by disturbed placental function or maternal malnutrition, prematurity, intrauterine and postnatal stress, intrauterine and postnatal overnutrition, as well as dietary dysbalances in postnatal life. This mini-review discusses critical developmental periods and long-term sequelae of renal programming in humans and presents studies examining the underlying mechanisms as well as interventional approaches to “re-program” renal susceptibility toward disease. Clinical manifestations of programmed kidney disease include arterial hypertension, proteinuria, aggravation of inflammatory glomerular disease, and loss of kidney function. Nephron number, regulation of the renin–angiotensin–aldosterone system, renal sodium transport, vasomotor and endothelial function, myogenic response, and tubuloglomerular feedback have been identified as being vulnerable to environmental factors. Oxidative stress levels, metabolic pathways, including insulin, leptin, steroids, and arachidonic acid, DNA methylation, and histone configuration may be significantly altered by adverse environmental conditions. Studies on re-programming interventions focused on dietary or anti-oxidative approaches so far. Further studies that broaden our understanding of renal programming mechanisms are needed to ultimately develop preventive strategies. Targeted re-programming interventions in animal models focusing on known mechanisms will contribute to new concepts which finally will have to be translated to human application

Ostomy Home Skills Program

Medline Plus

Full Text Available ... Trauma Quality Programs National Trauma Data Bank Trauma Quality Improvement Program Mentoring for Excellence in Trauma Surgery Advanced Trauma Life Support Verification, Review, and Consultation Program for Hospitals ...

Automatic programming for critical applications

Science.gov (United States)

Loganantharaj, Raj L.

1988-01-01

The important phases of a software life cycle include verification and maintenance. Usually, the execution performance is an expected requirement in a software development process. Unfortunately, the verification and the maintenance of programs are the time consuming and the frustrating aspects of software engineering. The verification cannot be waived for the programs used for critical applications such as, military, space, and nuclear plants. As a consequence, synthesis of programs from specifications, an alternative way of developing correct programs, is becoming popular. The definition, or what is understood by automatic programming, has been changed with our expectations. At present, the goal of automatic programming is the automation of programming process. Specifically, it means the application of artificial intelligence to software engineering in order to define techniques and create environments that help in the creation of high level programs. The automatic programming process may be divided into two phases: the problem acquisition phase and the program synthesis phase. In the problem acquisition phase, an informal specification of the problem is transformed into an unambiguous specification while in the program synthesis phase such a specification is further transformed into a concrete, executable program.

Economic Evaluation of a Comprehensive Teenage Pregnancy Prevention Program: Pilot Program

Science.gov (United States)

Rosenthal, Marjorie S.; Ross, Joseph S.; Bilodeau, RoseAnne; Richter, Rosemary S.; Palley, Jane E.; Bradley, Elizabeth H.

2011-01-01

Background Previous research has suggested that comprehensive teenage pregnancy prevention programs that address sexual education and life skills development and provide academic are effective in reducing births among enrolled teenagers. However, there have been limited data on costs and cost-effectiveness of such programs. Objectives To use a community-based participatory research approach, to develop estimates of the cost-benefit of the Pathways/Senderos Center, a comprehensive neighborhood-based program to prevent unintended pregnancies and promote positive development for adolescents. Methods Using data from 1997-2003, we conducted an in-time intervention analysis to determine program cost-benefit while teenagers were enrolled and then used an extrapolation analysis to estimate accyrred economibc benefits and cost-benefit up to age 30. Results The program operating costs totaled $3,228,152.59 and reduced the teenage childbearing rate from 94.10 to 40.00 per 1000 teenage females, averting $52,297.84 in total societal costs, with an economic benefit to society from program participation of $2,673,153.11. Therefore, total costs to society exceeded economic benefits by $559,677.05, or $1,599.08 per adolescent per year. In an extrapolation analysis, benefits to society exceed costs by $10,474.77 per adolescent per year by age 30 on average, with social benefits outweighing total social costs by age 20.1. Conclusions We estimate that this comprehensive teenage pregnancy prevention program would provide societal economic benefits once participants are young adults, suggesting the need to expand beyond pilot demonstrations and evaluate the long-range cost-effectiveness of similarly comprehensive programs when implemented more widely in high-risk neighborhoods. PMID:19896030

Economic evaluation of a comprehensive teenage pregnancy prevention program: pilot program.

Science.gov (United States)

Rosenthal, Marjorie S; Ross, Joseph S; Bilodeau, Roseanne; Richter, Rosemary S; Palley, Jane E; Bradley, Elizabeth H

2009-12-01

Previous research has suggested that comprehensive teenage pregnancy prevention programs that address sexual education and life skills development and provide academic support are effective in reducing births among enrolled teenagers. However, there have been limited data on the costs and cost effectiveness of such programs. The study used a community-based participatory research approach to develop estimates of the cost-benefit of the Pathways/Senderos Center, a comprehensive neighborhood-based program to prevent unintended pregnancies and promote positive development for adolescents. Using data from 1997-2003, an in-time intervention analysis was conducted to determine program cost-benefit while teenagers were enrolled; an extrapolation analysis was then used to estimate accrued economic benefits and cost-benefit up to age 30 years. The program operating costs totaled $3,228,152.59 and reduced the teenage childbearing rate from 94.10 to 40.00 per 1000 teenage girls, averting $52,297.84 in total societal costs, with an economic benefit to society from program participation of $2,673,153.11. Therefore, total costs to society exceeded economic benefits by $559,677.05, or $1599.08 per adolescent per year. In an extrapolation analysis, benefits to society exceed costs by $10,474.77 per adolescent per year by age 30 years on average, with social benefits outweighing total social costs by age 20.1 years. This comprehensive teenage pregnancy prevention program is estimated to provide societal economic benefits once participants are young adults, suggesting the need to expand beyond pilot demonstrations and evaluate the long-range cost effectiveness of similarly comprehensive programs when they are implemented more widely in high-risk neighborhoods.

Programs as Polypeptides.

Science.gov (United States)

Williams, Lance R

2016-01-01

Object-oriented combinator chemistry (OOCC) is an artificial chemistry with composition devices borrowed from object-oriented and functional programming languages. Actors in OOCC are embedded in space and subject to diffusion; since they are neither created nor destroyed, their mass is conserved. Actors use programs constructed from combinators to asynchronously update their own states and the states of other actors in their neighborhoods. The fact that programs and combinators are themselves reified as actors makes it possible to build programs that build programs from combinators of a few primitive types using asynchronous spatial processes that resemble chemistry as much as computation. To demonstrate this, OOCC is used to define a parallel, asynchronous, spatially distributed self-replicating system modeled in part on the living cell. Since interactions among its parts result in the construction of more of these same parts, the system is strongly constructive. The system's high normalized complexity is contrasted with that of a simple composome.

Fuel channel in-service inspection programs program design for maximum cost effectiveness

International Nuclear Information System (INIS)

Van den Brekel, N.C.

1995-01-01

Inspection is an integral part of fuel channel life management strategy. Inspection data is used to assess the state of reactor core integrity and provide the information necessary to optimize long term maintenance programs. This paper will provide an overview of the structured approach to developing fuel channel inspection programs within OHN. The inspection programs are designed to balance the resources utilized (cost, outage time, and dose expenditure) with the benefits provided by the inspection data obtained (improved knowledge of component status, degradation mechanisms and rates, etc..). The CANDU community has yet to have a fuel channel operate for a full 30 year design life. Since research programs can not fully simulate reactor operating conditions, inspections become an essential feature of the life management strategy as the components age. Inspection programs often include activities designed to develop predictive capability for long term fuel channel behaviour and provide early warning of changes in behaviour. It should be noted that although this paper addresses the design of fuel channel inspection programs, the basic principles presented can be applied to the design of inspection programs for any major power plant component or system. (author)

The SBIRT program matrix: a conceptual framework for program implementation and evaluation.

Science.gov (United States)

Del Boca, Frances K; McRee, Bonnie; Vendetti, Janice; Damon, Donna

2017-02-01

Screening, Brief Intervention and Referral to Treatment (SBIRT) is a comprehensive, integrated, public health approach to the delivery of services to those at risk for the adverse consequences of alcohol and other drug use, and for those with probable substance use disorders. Research on successful SBIRT implementation has lagged behind studies of efficacy and effectiveness. This paper (1) outlines a conceptual framework, the SBIRT Program Matrix, to guide implementation research and program evaluation and (2) specifies potential implementation outcomes. Overview and narrative description of the SBIRT Program Matrix. The SBIRT Program Matrix has five components, each of which includes multiple elements: SBIRT services; performance sites; provider attributes; patient/client populations; and management structure and activities. Implementation outcomes include program adoption, acceptability, appropriateness, feasibility, fidelity, costs, penetration, sustainability, service provision and grant compliance. The Screening, Brief Intervention and Referral to Treatment Program Matrix provides a template for identifying, classifying and organizing the naturally occurring commonalities and variations within and across SBIRT programs, and for investigating which variables are associated with implementation success and, ultimately, with treatment outcomes and other impacts. © 2017 Society for the Study of Addiction.

Stop smoking support programs

Science.gov (United States)

Smokeless tobacco - stop smoking programs; Stop smoking techniques; Smoking cessation programs; Smoking cessation techniques ... You can find out about smoking cessation programs from: Your ... Your employer Your local health department The National Cancer ...

National Transuranic Program Charter

International Nuclear Information System (INIS)

1994-10-01

The National Transuranic Program Plan and Charter describes the functional elements of the National TRU Program, organizational relationships, programmatic responsibilities, division of work scope among the various DOE organizations that comprise the program, and program baselines against which overall progress will be measured. The charter defines the authorities and responsibilities of various organizations involved in the management of TRU waste throughout the DOE complex

Coastal nonpoint pollution control program: Program development and approval guidance

International Nuclear Information System (INIS)

1993-01-01

The document, developed by NOAA and EPA, contains guidance for states in developing and implementing their coastal nonpoint pollutant source programs. It describes the requirements that must be met, including: the geographic scope of the program; the pollutant sources to be addressed; the types of management measures used; the establishment of critical areas; technical assistance, public participation, and administrative coordination; and, the process for program submission and Federal approval. The document also contains the criteria by which NOAA and EPA will review the states' submissions

77 FR 13530 - Funding for the Conservation Loan Program; Farm Loan Programs

Science.gov (United States)

2012-03-07

... DEPARTMENT OF AGRICULTURE Farm Service Agency Funding for the Conservation Loan Program; Farm Loan... Service Agency (FSA) now has funding for and is accepting guaranteed loan applications for the Conservation Loan (CL) Program. Due to a lack of program funding for direct CLs, direct loan applications are...

Training Accreditation Program

International Nuclear Information System (INIS)

1989-01-01

The Training Accreditation Program establishes the objectives and criteria against which DOE nuclear facility training is evaluated to determine its readiness for accreditation. Training programs are evaluated against the accreditation objectives and criteria by facility personnel during the initial self-evaluation process. From this self-evaluation, action plans are made by the contractor to address the scope of work necessary in order to upgrade any deficiencies noted. This scope of work must be formally documented in the Training Program Accreditation Plan. When reviewed and approved by the responsible Head of the Field Organization and cognizant Program Secretarial Office, EH-1 concurrence is obtained. This plan then becomes the document which guides accreditation efforts for the contractor

Energy research program 84

International Nuclear Information System (INIS)

1984-01-01

The energy research program 84 (EFP-84) is prepared by the Danish Ministry of Energy in order to continue the extension of the Danish energy research and development started through the former Trade Ministry's programs EM-1 (1976) and EM-2 (1978), and the Ministry of Energy's programs EFP-80, EFP-81, EFP-82 and EFP-83. The new program is a continuation of the activities in the period 1984-86 with a total budget of 112 mio. DKK. The program gives a brief description of background, principles, organization and financing, and a detailed description of each research area. (ln)

C++ how to program

CERN Document Server

Deitel, Paul J

2014-01-01

This best-selling comprehensive text is aimed at readers with little or no programming experience. It teaches programming by presenting the concepts in the context of full working programs and takes an early-objects approach. The authors emphasize achieving program clarity through structured and object-oriented programming, software reuse and component-oriented software construction. The Ninth Edition encourages students to connect computers to the community, using the Internet to solve problems and make a difference in our world. All content has been carefully fine-tuned in response to a team of distinguished academic and industry reviewers.

Energy research program 83

International Nuclear Information System (INIS)

1983-01-01

The energy research program 83 (EFP-83) is prepared by the Danish Ministry of Energy in order to continue the extension of the Danish energy research and development started through the former Trade Ministry's programs EM-1 (1976) and EM-2 (1978), and the Ministry of Energy's programs EFP-80, EFP-81 and EFP-82. The new program is a continuation of the activities in the period 1983-85 with a total budget of 111 mio. DKK. The program gives a brief description of background, principles, organization and financing, and a detailed description of each research area. (ln)

Energy research program 85

International Nuclear Information System (INIS)

1985-01-01

The energy research program 85 (EFP-85) is prepared by the Danish Ministry of Energy in order to continue the extension of the Danish energy research and development started through the former Trade Ministry's programs EM-1 (1976) and EM-2 (1978), and Ministry of Energy's programs EFP-80, EFP-81, EFP-82, EFP-83, and EFP-84. The new program is a continuation of the activities in the period 1985-87 with a total budget of 110 mio. DKK. The program gives a brief description of background, principles, organization and financing, and a detailed description of each research area. (ln)

Diabetes Prevention Program (DPP)

Science.gov (United States)

... Recruiting Patients & Families Consortia, Networks & Centers Reports & Planning Diabetes Prevention Program (DPP) The NIDDK-sponsored Diabetes Prevention ... Diabetes Prevention Program for those who are eligible. Diabetes Prevention Program (DPP) DPP Goal The DPP looked ...

Ostomy Home Skills Program

Medline Plus

Full Text Available ... Military Health System Strategic Partnership Military Health System Strategic Partnership About Excelsior Surgical Society ... Programs Quality Programs Overview About Quality Programs ACS Leadership in Quality ACS Leadership in Quality Setting the ...

New Production Reactors Program Plan

Energy Technology Data Exchange (ETDEWEB)

1990-12-01

Part I of this New Production Reactors (NPR) Program Plan: describes the policy basis of the NPR Program; describes the mission and objectives of the NPR Program; identifies the requirements that must be met in order to achieve the mission and objectives; and describes and assesses the technology and siting options that were considered, the Program's preferred strategy, and its rationale. The implementation strategy for the New Production Reactors Program has three functions: Linking the design, construction, operation, and maintenance of facilities to policies requirements, and the process for selecting options. The development of an implementation strategy ensures that activities and procedures are consistent with the rationale and analysis underlying the Program. Organization of the Program. The strategy establishes plans, organizational structure, procedures, a budget, and a schedule for carrying out the Program. By doing so, the strategy ensures the clear assignment of responsibility and accountability. Management and monitoring of the Program. Finally, the strategy provides a basis for monitoring the Program so that technological, cost, and scheduling issues can be addressed when they arise as the Program proceeds. Like the rest of the Program Plan, the Implementation Strategy is a living document and will be periodically revised to reflect both progress made in the Program and adjustments in plans and policies as they are made. 21 figs., 5 tabs.

New Production Reactors Program Plan

International Nuclear Information System (INIS)

1990-12-01

Part I of this New Production Reactors (NPR) Program Plan: describes the policy basis of the NPR Program; describes the mission and objectives of the NPR Program; identifies the requirements that must be met in order to achieve the mission and objectives; and describes and assesses the technology and siting options that were considered, the Program's preferred strategy, and its rationale. The implementation strategy for the New Production Reactors Program has three functions: Linking the design, construction, operation, and maintenance of facilities to policies requirements, and the process for selecting options. The development of an implementation strategy ensures that activities and procedures are consistent with the rationale and analysis underlying the Program. Organization of the Program. The strategy establishes plans, organizational structure, procedures, a budget, and a schedule for carrying out the Program. By doing so, the strategy ensures the clear assignment of responsibility and accountability. Management and monitoring of the Program. Finally, the strategy provides a basis for monitoring the Program so that technological, cost, and scheduling issues can be addressed when they arise as the Program proceeds. Like the rest of the Program Plan, the Implementation Strategy is a living document and will be periodically revised to reflect both progress made in the Program and adjustments in plans and policies as they are made. 21 figs., 5 tabs

State Energy Program Operations Manual

Energy Technology Data Exchange (ETDEWEB)

Office of Building Technology, State and Community Programs

1999-03-17

The State Energy Program Operations Manual is a reference tool for the states and the program officials at the U.S. Department of Energy's Office of Building Technology, State and Community Programs and Regional Support Offices as well as State Energy Offices. The Manual contains information needed to apply for and administer the State Energy Program, including program history, application rules and requirements, and program administration and monitoring requirements.

Higher-Order Program Generation

DEFF Research Database (Denmark)

Rhiger, Morten

for OCaml, a dialect of ML, that provides run-time code generation for OCaml programs. We apply these byte-code combinators in semantics-directed compilation for an imperative language and in run-time specialization using type-directed partial evaluation. Finally, we present an approach to compiling goal......This dissertation addresses the challenges of embedding programming languages, specializing generic programs to specific parameters, and generating specialized instances of programs directly as executable code. Our main tools are higher-order programming techniques and automatic program generation....... It is our thesis that they synergize well in the development of customizable software. Recent research on domain-specific languages propose to embed them into existing general-purpose languages. Typed higher-order languages have proven especially useful as meta languages because they provide a rich...

Computer Program Newsletter No. 7

International Nuclear Information System (INIS)

Magnuson, W.G. Jr.

1982-09-01

This issue of the Computer Program Newsletter updates an earlier newsletter (Number 2, September 1979) and focuses on electrical network analysis computer programs. In particular, five network analysis programs (SCEPTRE, SPICE2, NET2, CALAHAN, and EMTP) will be described. The objective of this newsletter will be to provide a very brief description of the input syntax and semantics for each program, highlight their strong and weak points, illustrate how the programs are run at Lawrence Livermore National Laboratory using the Octopus computer network, and present examples of input for each of the programs to illustrate some of the features of each program. In a sense, this newsletter can be used as a quick reference guide to the programs

On termination of meta-programs

NARCIS (Netherlands)

Serebrenik, A.; De Schreye, D.; Nieuwenhuis, R.; Voronkov, A.

2001-01-01

The term meta-programming refers to the ability of writing programs that have other programs as data and exploit their semantics [4]. The choice of logic programming as a basis for meta-programming offers a number of practical and theoretical advantages. One of them is the possibility of tackling

Attitudes of program directors toward women in pediatric dentistry training programs.

Science.gov (United States)

Seale, N S; Waggoner, W F

1992-01-01

The number of women entering pediatric dentistry graduate programs is increasing. A formal survey was conducted in the fall of 1990 to determine what impact, if any, this increase is having on the programs. The survey sample consisted of the 57 pediatric dentistry graduate program directors from the United States and Canada. The survey form included program data about gender distribution in the current and previous classes, and female faculty distribution within the programs. The survey requested information about the attitudes of various groups of individuals who interacted with the residents relative to the gender of the resident and again, relative to whether the resident was pregnant. Inquiry was made concerning maternity leave policies and selected treatment scenarios involving pregnant residents. Finally, questions were asked about motivational factors, personal priorities, and policy change for female vs. male residents. Fifty forms were returned for a return rate of 88%. The 48 forms analyzed revealed that 52% of current classes are female and 51% of applicants for 1991 were female. Women comprise 23% of full-time and 26% of part-time faculty. There was no single issue perceived by program directors as a group to be a significant concern or problem relating to gender. Program directors would consider removing pregnant females from contact with combative patients (83%) and environmental hazards (85%), but fewer would consider removing them from contact with for HIV+ or Hb+ patients.

Multi-dimensional Rankings, Program Termination, and Complexity Bounds of Flowchart Programs

Science.gov (United States)

Alias, Christophe; Darte, Alain; Feautrier, Paul; Gonnord, Laure

Proving the termination of a flowchart program can be done by exhibiting a ranking function, i.e., a function from the program states to a well-founded set, which strictly decreases at each program step. A standard method to automatically generate such a function is to compute invariants for each program point and to search for a ranking in a restricted class of functions that can be handled with linear programming techniques. Previous algorithms based on affine rankings either are applicable only to simple loops (i.e., single-node flowcharts) and rely on enumeration, or are not complete in the sense that they are not guaranteed to find a ranking in the class of functions they consider, if one exists. Our first contribution is to propose an efficient algorithm to compute ranking functions: It can handle flowcharts of arbitrary structure, the class of candidate rankings it explores is larger, and our method, although greedy, is provably complete. Our second contribution is to show how to use the ranking functions we generate to get upper bounds for the computational complexity (number of transitions) of the source program. This estimate is a polynomial, which means that we can handle programs with more than linear complexity. We applied the method on a collection of test cases from the literature. We also show the links and differences with previous techniques based on the insertion of counters.

THE ESTABLISHMENT OF A U.S. SUPPORT PROGRAM INTERSHIP PROGRAM

International Nuclear Information System (INIS)

PEPPER, S.E.

2003-01-01

In 2002, the U.S. Support Program to IAEA Safeguards established a program of one-year paid internships with the IAEA Department of Safeguards for students and recent graduates. Six interns are currently working with the IAEA in software development and information collection activities. The program is administered through the International Safeguards Project Office (ISPO) at Brookhaven National Laboratory (BNL). Software development assignments were considered to be most feasible because of the considerable abilities of many computer science students after a few years' education. Candidates in information science were also recruited because of an existing internship program managed by the Monterey Institute of International Studies. ISPO recruited students from US. colleges and other sources. Applications were collected and provided to the IAEA for review and selection. SGIT then identified the best applicants and, after confirming their intention to accept the position, tailored assignments based on their qualifications. Before the assignments started, ISPO conducted an orientation to provide the interns with information to ease their transition into working with the IAEA and living in Vienna. Four interns began their assignments in software development in June 2002 and two others began their assignments in information collection in July and August. The IAEA, the interns, and the Subgroup on Safeguards Technical Support have found the assignments to be beneficial. The internship program provides additional staff to the IAEA at low cost to the USSP, introduces young professionals to careers in the nuclear industry and international civil service, and provides the IAEA access to U.S. academic institutions. In 2003, the program will be expanded to include engineering and technical writing in support of the Division of Safeguards Technical Services. The paper will discuss the recruitment and selection of interns and the administration of the program

76 FR 54599 - Medicare Program; Medicare Advantage and Prescription Drug Benefit Programs

Science.gov (United States)

2011-09-01

...), prescription drug benefit program (Part D) and section 1876 cost plans including conforming changes to the MA... accounts (MSA) plans, cost-sharing for dual-eligible enrollees in the MA program and prescription drug pricing, coverage, and payment processes in the Part D program, and requirements governing the marketing...

Evaluating Effectiveness of Pair Programming as a Teaching Tool in Programming Courses

Science.gov (United States)

Faja, Silvana

2014-01-01

This study investigates the effectiveness of pair programming on student learning and satisfaction in introductory programming courses. Pair programming, used in the industry as a practice of an agile development method, can be adopted in classroom settings to encourage peer learning, increase students' social skills, and enhance student…

76 FR 39006 - Medicare Program; Hospital Inpatient Value-Based Purchasing Program; Correction

Science.gov (United States)

2011-07-05

... and 480 [CMS-3239-CN] RIN 0938-AQ55 Medicare Program; Hospital Inpatient Value-Based Purchasing... Value-Based Purchasing Program.'' DATES: Effective Date: These corrections are effective on July 1, 2011... for the hospital value-based purchasing program. Therefore, in section III. 6. and 7. of this notice...

Tank Focus Area Pretreatment Program. FY 1995 Program Management Plan

International Nuclear Information System (INIS)

Morrison, M.I.; McGinnis, C.P.; Wilkenson, W.T.; Hunt, R.D.

1995-02-01

This program management plan (PMP) describes the FY 1995 project plans for the Pretreatment Program of the Tank Focus Area. The Tank Focus Area is one of five areas of environmental concerns originally identified by the Deputy Assistant Secretary for Technology Development (EM-50). Projects in the Tank Focus Area relate to the remediation of liquid waste stored in underground storage tanks at various US Department of Energy sites. The Pretreatment Program is an organizational unit performing work within the Tank Focus Area. The function of the Pretreatment Program is to develop, test, evaluate, and demonstrate new technologies, with emphasis on separations. The 11 Pretreatment Program projects for FY 1995 are (1) Cesium Extraction Testing, (2) Comprehensive Supernate Treatment, (3) Hot Cell Studies, (4) Cesium Removal Demonstration, (5) Out-of-Tank Evaporator Demonstration, (6) Crossflow Filtration, (7) Technical Interchange with CEA, (8) TRUEX Applications, (9) NAC/NAG Process Studies (conducted at Oak Ridge National Laboratory), (10) NAC/NAG Process and Waste Form Studies (conducted at Florida International University), and (11) Program Management. Section 2 of this PMP contains a separate subsection for each FY 1995 project. A brief description of the project, a schedule of major milestones, and a breakdown of costs are provided for each project. The PMP also contains sections that describe the project controls that are in place. Quality assurance, document control, the project management system, and the management organization are described in these sections

A cross-cultural mentoring program

Energy Technology Data Exchange (ETDEWEB)

Huang-Nissen, S.; Myers, R.Y.

1995-04-01

This report summarized the results of the pilot Cross-Cultural Mentoring Program at Lawrence Livermore National Laboratory, from the inception of the program idea through its implementation and assessment. It discusses the benefits of mentoring, the origins of the program, program design and implementation, program assessment, and conclusions and recommendations.

Integrating Professional Development into STEM Graduate Programs: Student-Centered Programs for Career Preparation

Science.gov (United States)

Lautz, L.; McCay, D.; Driscoll, C. T.; Glas, R. L.; Gutchess, K. M.; Johnson, A.; Millard, G.

2017-12-01

Recognizing that over half of STEM Ph.D. graduates are finding work outside of academia, a new, NSF-funded program at Syracuse University, EMPOWER (or Education Model Program on Water-Energy Research) is encouraging its graduate students to take ownership of their graduate program and design it to meet their anticipated needs. Launched in 2016, EMPOWER's goal is to prepare graduate students for careers in the water-energy field by offering targeted workshops, professional training coursework, a career capstone experience, a professional development mini-grant program, and an interdisciplinary "foundations" seminar. Through regular student feedback and program evaluation, EMPOWER has learned some important lessons this first year: career options and graduate students' interests are diverse, requiring individualized programs designed to meet the needs of prospective employers and employees; students need exposure to the range of careers in their field to provide a roadmap for designing their own graduate school experience; effective programs nurture a culture that values professional development thereby giving students permission to pursue career paths and professional development opportunities that meet their own needs and interests; and existing university resources support the effective and efficient integration of professional development activities into graduate programs. Many of the positive outcomes experienced by EMPOWER students may be achieved in departmental graduate programs with small changes to their graduate curricula.

Supervisor's role in training programs as a manager of learning program

Directory of Open Access Journals (Sweden)

2011-06-01

Full Text Available According to the training literature, a supervisor's role in training programs has two major elements: supervisor support and supervisor communication. The ability of supervisors to play effective roles in training programs may increase employees' motivation to learn. The nature of this relationship is interesting, but the role of supervisor's role as a predicting variable is less emphasized in a training program models. Therefore, this study was conducted to examine the effect of supervisor's role in training programs on motivation to learn using 152 usable questionnaires gathered from non-academic employees who have worked in a technological based public university, Malaysia. The outcomes of stepwise regression analysis showed that the supervisor support and supervisor communication significantly associated with motivation to learn. Statistically, this result demonstrates that supervisor's role in training programs does act as an important predictor of motivation to learn in the organizational sample. In addition, discussion, implication and conclusion are elaborated.

Ostomy Home Skills Program

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Full Text Available ... Accreditation Program for Breast Centers About NAPBC Accreditation Education NAPBC Standards Cancer Programs News Quality in Geriatric ... Continuous Certification Requirements SSR Login MIPS Resources and Education Quality and Safety Conference Trauma Trauma Programs Trauma ...

Ostomy Home Skills Program

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Ostomy Home Skills Program

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Full Text Available ... and Safety Inspiring Quality Initiative Resources Continuous Quality Improvement ACS Clinical Scholars in Residence AHRQ Safety Program ... Enrollment Webinars ACS NSQIP ACS National Surgical Quality Improvement Program ACS National Surgical Quality Improvement Program About ...

Ada Linear-Algebra Program

Science.gov (United States)

Klumpp, A. R.; Lawson, C. L.

1988-01-01

Routines provided for common scalar, vector, matrix, and quaternion operations. Computer program extends Ada programming language to include linear-algebra capabilities similar to HAS/S programming language. Designed for such avionics applications as software for Space Station.

Ostomy Home Skills Program

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Full Text Available ... Policy Updates Selected Research Findings Quality Program Initiatives Communications to the Profession Advocacy Advocacy Overview Quality Payment Program QPP Resource Center QPP Resource Center 2018 Information 2017 Information Program Rules Surgeon Specific Registry Metabolic ...

Ostomy Home Skills Program

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Full Text Available ... Membership Directory 2017 Annual Meeting 2016 Annual Meeting Women's Committee Mentorship Program Outside Activities ACS Archives Contact ... Accreditation Program for Breast Centers About NAPBC Accreditation Education NAPBC Standards Cancer Programs News Quality in Geriatric ...

PNNL FY2005 DOE Voluntary Protection Program (VPP) Program Evaluation

Energy Technology Data Exchange (ETDEWEB)

Wright, Patrick A.; Madson, Vernon J.; Isern, Nancy G.; Haney, Janice M.; Fisher, Julie A.; Goheen, Steven C.; Gulley, Susan E.; Reck, John J.; Collins, Drue A.; Tinker, Mike R.; Walker, Landon A.; Wynn, Clifford L.

2005-01-31

This document reports the results of the FY 2005 PNNL VPP Program Evaluation, which is a self-assessment of the operational and programmatic performance of the Laboratory related to worker safety and health. The report was compiled by a team of worker representatives and safety professionals who evaluated the Laboratory's worker safety and health programs on the basis of DOE-VPP criteria. The principle elements of DOE's VPP program are: Management Leadership, Employee Involvement, Worksite Analysis, Hazard Prevention and Control, and Safety and Health Training.

Report of the Integrated Program Planning Activity for the DOE Fusion Energy Sciences Program

International Nuclear Information System (INIS)

None

2000-01-01

This report of the Integrated Program Planning Activity (IPPA) has been prepared in response to a recommendation by the Secretary of Energy Advisory Board that, ''Given the complex nature of the fusion effort, an integrated program planning process is an absolute necessity.'' We, therefore, undertook this activity in order to integrate the various elements of the program, to improve communication and performance accountability across the program, and to show the inter-connectedness and inter-dependency of the diverse parts of the national fusion energy sciences program. This report is based on the September 1999 Fusion Energy Sciences Advisory Committee's (FESAC) report ''Priorities and Balance within the Fusion Energy Sciences Program''. In its December 5,2000, letter to the Director of the Office of Science, the FESAC has reaffirmed the validity of the September 1999 report and stated that the IPPA presents a framework and process to guide the achievement of the 5-year goals listed in the 1999 report. The National Research Council's (NRC) Fusion Assessment Committee draft final report ''An Assessment of the Department of Energy's Office of Fusion Energy Sciences Program'', reviewing the quality of the science in the program, was made available after the IPPA report had been completed. The IPPA report is, nevertheless, consistent with the recommendations in the NRC report. In addition to program goals and the related 5-year, 10-year, and 15-year objectives, this report elaborates on the scientific issues associated with each of these objectives. The report also makes clear the relationships among the various program elements, and cites these relationships as the reason why integrated program planning is essential. In particular, while focusing on the science conducted by the program, the report addresses the important balances between the science and energy goals of the program, between the MFE and IFE approaches, and between the domestic and international aspects

Laboratory Cooperative Program: an assessment

International Nuclear Information System (INIS)

1979-11-01

The Laboratory Cooperative Program (Lab Coop Program) was initiated by the US AEC over 20 years ago to promote the transfer of technical information from the national laboratories to the academic community utilizing the facilities and staff capabilities of the labs. Under the AEC, ERDA and DOE, the goals of the program have broadened gradually. Therefore, the program was examined to determine the extent to which it contributes to the current objectives of the DOE and to develop recommendations for any program changes. The assessment of the Lab Coop Program was based on a combination of review of program activity data and publications, review of general information regarding laboratory operations, and extensive interviews. The major findings of this evaluation were that: the program lacks a clear statement of purpose; program plans, priorities, and procedures are not explicit and operations tend to follow historical patterns; and the program is generally accepted as beneficial, but its benefits are difficult to quantify. It is recommended that the focus of the Lab Coop Program be limited and clearly defined, that performance plans be developed and measured against accomplishments, and that a national informational effort be initiated

Developmental Programming of Renal Function and Re-Programming Approaches

Directory of Open Access Journals (Sweden)

Eva Nüsken

2018-02-01

Full Text Available Chronic kidney disease affects more than 10% of the population. Programming studies have examined the interrelationship between environmental factors in early life and differences in morbidity and mortality between individuals. A number of important principles has been identified, namely permanent structural modifications of organs and cells, long-lasting adjustments of endocrine regulatory circuits, as well as altered gene transcription. Risk factors include intrauterine deficiencies by disturbed placental function or maternal malnutrition, prematurity, intrauterine and postnatal stress, intrauterine and postnatal overnutrition, as well as dietary dysbalances in postnatal life. This mini-review discusses critical developmental periods and long-term sequelae of renal programming in humans and presents studies examining the underlying mechanisms as well as interventional approaches to “re-program” renal susceptibility toward disease. Clinical manifestations of programmed kidney disease include arterial hypertension, proteinuria, aggravation of inflammatory glomerular disease, and loss of kidney function. Nephron number, regulation of the renin–angiotensin–aldosterone system, renal sodium transport, vasomotor and endothelial function, myogenic response, and tubuloglomerular feedback have been identified as being vulnerable to environmental factors. Oxidative stress levels, metabolic pathways, including insulin, leptin, steroids, and arachidonic acid, DNA methylation, and histone configuration may be significantly altered by adverse environmental conditions. Studies on re-programming interventions focused on dietary or anti-oxidative approaches so far. Further studies that broaden our understanding of renal programming mechanisms are needed to ultimately develop preventive strategies. Targeted re-programming interventions in animal models focusing on known mechanisms will contribute to new concepts which finally will have to be translated

75 FR 48815 - Medicaid Program and Children's Health Insurance Program (CHIP); Revisions to the Medicaid...

Science.gov (United States)

2010-08-11

... Parts 431, 447, and 457 Medicaid Program and Children's Health Insurance Program (CHIP); Revisions to... 431, 447, and 457 [CMS-6150-F] RIN 0938-AP69 Medicaid Program and Children's Health Insurance Program... final rule implements provisions from the Children's Health Insurance Program Reauthorization Act of...

PROGRAMMING IS GOOD FOR CHILDREN? A CRITICAL VIEW ABOUT TEACHING PROGRAMMING IN SCHOOLS

Directory of Open Access Journals (Sweden)

Wendell Bento Geraldes

2014-12-01

Full Text Available This article presents reflections on teaching programming in schools and the positive and negative impact of this new methodology today. The study also discusses the initiatives relating to teaching programming in schools, considering also the opinion of experts on the subject. The following questions are addressed: Is it good for children to learn to program computers in schools? Can all people learn to program computers? What is the importance of learning for today's society? The pros and cons regarding teaching programming in schools will be discussed in search of answers to these questions.

Quality assurance program plan for the Reactor Research Experiment Programs (RREP)

International Nuclear Information System (INIS)

Pipher, D.G.

1982-05-01

This document describes the Quality Assurance Program plans which will be applied to tasks on Reactor Research Experiments performed on Sandia National Laboratories' reactors. The program provides for individual project or experiment quality plan development and allows for reasonable plan flexibility and maximum plan visibility. Various controls and requirements in this program plan are considered mandatory on all features which are identified as important to public health and safety (Level I). It is the intent of this document that the Quality Assurance program comprise those elements which will provide adequate assurance that all components, equipment, and systems of the experiments will perform as designed, and hence prevent delays and costs due to rejections or failures

Department of Energy hazardous waste remedial actions program: Quality assurance program

International Nuclear Information System (INIS)

Horne, T.E.

1988-01-01

This paper describes the Quality Assurance Program developed for the Hazardous Waste Remedial Actions Program Support Contractor Office (HAZWRAP SCO). Key topics discussed include an overview of the HAZWRAP SCO mission and organization, the basic quality assurance program requirements and the requirements for the control of quality for the Department of Energy and Work for Others hazardous waste management programs, and the role of ensuring quality through the project team concept for the management of remedial response actions. The paper focuses on planning for quality assurance for this remedial waste management process from preliminary assessments of remedial sites to feasibility studies. Some observations concerning the control of quality during the implementation of remedial actions are presented. (2 refs.)

Programming in biomolecular computation

DEFF Research Database (Denmark)

Hartmann, Lars Røeboe; Jones, Neil; Simonsen, Jakob Grue

2011-01-01

Our goal is to provide a top-down approach to biomolecular computation. In spite of widespread discussion about connections between biology and computation, one question seems notable by its absence: Where are the programs? We identify a number of common features in programming that seem...... conspicuously absent from the literature on biomolecular computing; to partially redress this absence, we introduce a model of computation that is evidently programmable, by programs reminiscent of low-level computer machine code; and at the same time biologically plausible: its functioning is defined...... by a single and relatively small set of chemical-like reaction rules. Further properties: the model is stored-program: programs are the same as data, so programs are not only executable, but are also compilable and interpretable. It is universal: all computable functions can be computed (in natural ways...

The art of multiprocessor programming

CERN Document Server

Herlihy, Maurice

2012-01-01

Revised and updated with improvements conceived in parallel programming courses, The Art of Multiprocessor Programming is an authoritative guide to multicore programming. It introduces a higher level set of software development skills than that needed for efficient single-core programming. This book provides comprehensive coverage of the new principles, algorithms, and tools necessary for effective multiprocessor programming. Students and professionals alike will benefit from thorough coverage of key multiprocessor programming issues. This revised edition incorporates much-demanded updates t

Energy research program 86

International Nuclear Information System (INIS)

1986-01-01

The energy research program 86 (EFP-86) is prepared by the Danish Ministry of Energy in order to continue the extension of the Danish energy research and development started through the former Trade Ministry's programs EM-1 (1976) and EM-2 (1978), and the Ministry of Energy's programs EFP-80, EFP-81, EFP-82, EFP-83, EFP-84, and EFP-85. The new program is a continuation of the activities in the period 1986-88 with a total budget of 116 mio. DKK. The program gives a brief description of background, principles, organization and financing, and a detailed description of each research area. (ln)

Ostomy Home Skills Program

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Full Text Available ... Trauma Programs About Trauma Programs Violence Prevention BleedingControl.org Trauma Quality Programs National Trauma Data Bank Trauma ... 5000 (F) 312-202-5001 (E) postmaster@facs.org Copyright © 1996-2018 by the American College of ...

Ostomy Home Skills Program

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Full Text Available ... Quality and Safety Inspiring Quality Initiative Resources Continuous Quality Improvement ACS Clinical Scholars in Residence AHRQ Safety Program ... ISCR Enrollment Webinars ACS NSQIP ACS National Surgical Quality Improvement Program ACS National Surgical Quality Improvement Program About ...

Building Technologies Program Multi-Year Program Plan Research and Development 2008

Energy Technology Data Exchange (ETDEWEB)

None, None

2008-01-01

Building Technologies Program Multi-Year Program Plan 2008 for research and development, including residential and commercial integration, lighting, HVAC and water heating, envelope, windows, and analysis tools.

Reactive Programming in Java

CERN Document Server

CERN. Geneva

2017-01-01

Reactive Programming in gaining a lot of excitement. Many libraries, tools, and frameworks are beginning to make use of reactive libraries. Besides, applications dealing with big data or high frequency data can benefit from this programming paradigm. Come to this presentation to learn about what reactive programming is, what kind of problems it solves, how it solves them. We will take an example oriented approach to learning the programming model and the abstraction.

Program Aplikasi Optimalisasi Perencanaan Produksi Dengan Metode Goal Programming (Kasus Cv. G

Directory of Open Access Journals (Sweden)

Zahedi Zahedi

2010-06-01

Full Text Available Indonesia today is still a State agrarian. Thus agriculture in Indonesia always get more attention by the government. Many government programs are rolled out to support the agricultural development of Indonesia, and one of them is the fertilizer subsidy to farmers Indonesia. Fertilizer itself is very useful to nourish the soil and provide vitamins to the plants, thus increasing the amount of production generated. Therefore it is necessary fertilizer for agriculture in Indonesia. CV. G company is one of the companies that produce fertilizer. Fertilizer produced by this company is an organic fertilizer and inorganic fertilizer. Problems faced by these companies are in planning the production of fertilizer produced. Companies want to optimize the amount of production of each fertilizer, maximize revenues, minimize production costs, and simultaneously optimize the working hours of employees. To meet these objectives, this research created program package using goal programming methods. Goal programming method can provide an effective solution / equilibrium toward the goals set, and the calculation will be assisted by using the Simplex method that has been modified. This method is an extension of linear programming, linear programming which can only solve problems with a single goal while the goal programming to solve problems with multiple objectives.

Doctoral Study Programs in Social Work at HBCUs: Origin and Program Development

Science.gov (United States)

Aubrey, Hal; Jordan, Tina; Stevenson, Andre P.; Boss-Victoria, Rena; Haynes, James; Estreet, Anthony; Smith, Jahmaine; Cameron, Elijah; Williams, Quotasze

2016-01-01

Fisk University began the genesis of HBCU graduate programs in 1880. During the next fifty years, several other HBCUs established graduate programs. That group included Lincoln, Howard, and Morgan State. However, only Lincoln University established a PhD program. The primary goal of this paper is to provide a historical perspective regarding the…

Using program impact pathways to understand and improve program delivery, utilization, and potential for impact of Helen Keller International's homestead food production program in Cambodia.

Science.gov (United States)

Olney, Deanna K; Vicheka, Sao; Kro, Meng; Chakriya, Chhom; Kroeun, Hou; Hoing, Ly Sok; Talukder, Aminzzaman; Quinn, Victoria; Iannotti, Lora; Becker, Elisabeth; Roopnaraine, Terry

2013-06-01

Evidence of the impact of homestead food production programs on nutrition outcomes such as anemia and growth is scant. In the absence of information on program impact pathways, it is difficult to understand why these programs, which have been successful in increasing intake of micronutrient-rich foods, have had such limited documented impact on nutrition outcomes. To conduct a process evaluation of Helen Keller International's (HKI's) homestead food production program in Cambodia to assess whether the program was operating as planned (in terms of design, delivery, and utilization) and to identify ways in which the program might need to be strengthened in order to increase its potential for impact. A program theory framework, which laid out the primary components along the hypothesized program impact pathways, was developed in collaboration with HKI and used to design the research. Semistructured interviews and focus group discussions with program beneficiaries (n = 36 and 12, respectively), nonbeneficiaries (n = 12), and program implementers (n = 17 and 2, respectively) and observations of key program delivery points, including health and nutrition training sessions (n = 6), village model farms (n = 6), and household gardens of beneficiaries (n = 36) and nonbeneficiaries (n = 12), were conducted to assess the delivery and utilization of the primary program components along the impact pathways. The majority of program components were being delivered and utilized as planned. However, challenges with some of the key components posited to improve outcomes such as anemia and growth were noted. Among these were a gap in the expected pathway from poultry production to increased intake of eggs and poultry meat, and some weaknesses in the delivery of the health and nutrition training sessions and related improvements in knowledge among the village health volunteers and beneficiaries. Although the program has been successful in delivering the majority of the program

Meteorological Monitoring Program

International Nuclear Information System (INIS)

Hancock, H.A. Jr.; Parker, M.J.; Addis, R.P.

1994-01-01

The purpose of this technical report is to provide a comprehensive, detailed overview of the meteorological monitoring program at the Savannah River Site (SRS) near Aiken, South Carolina. The principle function of the program is to provide current, accurate meteorological data as input for calculating the transport and diffusion of any unplanned release of an atmospheric pollutant. The report is recommended for meteorologists, technicians, or any personnel who require an in-depth understanding of the meteorological monitoring program

Meteorological Monitoring Program

Energy Technology Data Exchange (ETDEWEB)

Hancock, H.A. Jr. [ed.; Parker, M.J.; Addis, R.P.

1994-09-01

The purpose of this technical report is to provide a comprehensive, detailed overview of the meteorological monitoring program at the Savannah River Site (SRS) near Aiken, South Carolina. The principle function of the program is to provide current, accurate meteorological data as input for calculating the transport and diffusion of any unplanned release of an atmospheric pollutant. The report is recommended for meteorologists, technicians, or any personnel who require an in-depth understanding of the meteorological monitoring program.

Integer programming

CERN Document Server

Conforti, Michele; Zambelli, Giacomo

2014-01-01

This book is an elegant and rigorous presentation of integer programming, exposing the subject’s mathematical depth and broad applicability. Special attention is given to the theory behind the algorithms used in state-of-the-art solvers. An abundance of concrete examples and exercises of both theoretical and real-world interest explore the wide range of applications and ramifications of the theory. Each chapter is accompanied by an expertly informed guide to the literature and special topics, rounding out the reader’s understanding and serving as a gateway to deeper study. Key topics include: formulations polyhedral theory cutting planes decomposition enumeration semidefinite relaxations Written by renowned experts in integer programming and combinatorial optimization, Integer Programming is destined to become an essential text in the field.

Ecosystem Analysis Program

International Nuclear Information System (INIS)

Burgess, R.L.

1978-01-01

Progress is reported on the following research programs: analysis and modeling of ecosystems; EDFB/IBP data center; biome analysis studies; land/water interaction studies; and computer programs for development of models

Ostomy Home Skills Program

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The 3-D diffusion program XYZ-MUGDI with data-analyzing program XYZFF

International Nuclear Information System (INIS)

Siewers, H.

1976-01-01

The XYZ-MUGDI program solves boudary value and eigenvalue problems for three-dimensional heterogeneous configurations in X, Y, Z-geometry for a maximum of four energy groups. The program XYZFF is a program for analyzing data; with it especially the average and maximum form factors for arbitrary coarse-meshed networks can be evaluated. (orig.) [de

25 CFR 170.804 - How is BIA's Road Maintenance Program related to the IRR Program?

Science.gov (United States)

2010-04-01

... 25 Indians 1 2010-04-01 2010-04-01 false How is BIA's Road Maintenance Program related to the IRR... WATER INDIAN RESERVATION ROADS PROGRAM BIA Road Maintenance § 170.804 How is BIA's Road Maintenance Program related to the IRR Program? The following chart illustrates how BIA's Road Maintenance Program is...

A Qualitative Program Evaluation of a Structured Leadership Mentoring Program at a Large Aerospace Corporation

Science.gov (United States)

Teller, Romney P.

2011-01-01

The researcher utilized a qualitative approach to conduct a program evaluation of the organization where he is employed. The study intended to serve as a program evaluation for the structured in-house mentoring program at a large aerospace corporation (A-Corp). This program evaluation clarified areas in which the current mentoring program is…

The program success story: a valuable tool for program evaluation.

Science.gov (United States)

Lavinghouze, Rene; Price, Ann Webb; Smith, Kisha-Ann

2007-10-01

Success stories are evaluation tools that have been used by professionals across disciplines for quite some time. They are also proving to be useful in promoting health programs and their accomplishments. The increasing popularity of success stories is due to the innovative and effective way that they increase a program's visibility, while engaging potential participants, partners, and funders in public health efforts. From the community level to the federal level, program administrators are using success stories as vehicles for celebrating achievements, sharing challenges, and communicating lessons learned. Success stories are an effective means to move beyond the numbers and connect to readers-with a cause they can relate to and want to join. This article defines success stories and provides an overview of several types of story formats, how success stories can be systematically collected, and how they are used to communicate program success.

Programs, interfaces and components

NARCIS (Netherlands)

Bergstra, J.A.; Loots, M.E.

The jump instruction is considered essential for an adequate theoretical understanding of imperative sequential programming. Using atomic actions and tests as a basis we outline an algebra of programs, denoted PGA, which captures the crux of sequential programming. PGA provides an ontology for

Trends in Multicultural Programming.

Science.gov (United States)

Mylopoulos, Chryss

1985-01-01

Outlines basic principles and philosophy behind library multicultural programs and provides brief overview of development of such programs in Canadian libraries. Programing themes (cultural identity, contribution of ethnocultural groups to Canadian society, interpretation of multiculturalism as social policy) and suggestions for integrating…

HIV Programs for Sex Workers: Lessons and Challenges for Developing and Delivering Programs.

Science.gov (United States)

Wilson, David

2015-06-01

There is evidence that HIV prevention programs for sex workers, especially female sex workers, are cost-effective in several contexts, including many western countries, Thailand, India, the Democratic Republic of Congo, Kenya, and Zimbabwe. The evidence that sex worker HIV prevention programs work must not inspire complacency but rather a renewed effort to expand, intensify, and maximize their impact. The PLOS Collection "Focus on Delivery and Scale: Achieving HIV Impact with Sex Workers" highlights major challenges to scaling-up sex worker HIV prevention programs, noting the following: sex worker HIV prevention programs are insufficiently guided by understanding of epidemic transmission dynamics, situation analyses, and programmatic mapping; sex worker HIV and sexually transmitted infection services receive limited domestic financing in many countries; many sex worker HIV prevention programs are inadequately codified to ensure consistency and quality; and many sex worker HIV prevention programs have not evolved adequately to address informal sex workers, male and transgender sex workers, and mobile- and internet-based sex workers. Based on the wider collection of papers, this article presents three major clusters of recommendations: (i) HIV programs focused on sex workers should be prioritized, developed, and implemented based on robust evidence; (ii) national political will and increased funding are needed to increase coverage of effective sex worker HIV prevention programs in low and middle income countries; and (iii) comprehensive, integrated, and rapidly evolving HIV programs are needed to ensure equitable access to health services for individuals involved in all forms of sex work.

Photovoltaic systems. Program summary

Energy Technology Data Exchange (ETDEWEB)

None

1978-12-01

Each of the Department of Energy's Photovoltaic Systems Program projects funded and/or in existence during fiscal year 1978 (October 1, 1977 through September 30, 1978) are described. The project sheets list the contractor, principal investigator, and contract number and funding and summarize the programs and status. The program is divided into various elements: program assessment and integration, research and advanced development, technology development, system definition and development, system application experiments, and standards and performance criteria. (WHK)

Putting program evaluation into practice: enhancing the Girls Just Wanna Have Fun program.

Science.gov (United States)

Bean, Corliss N; Kendellen, Kelsey; Halsall, Tanya; Forneris, Tanya

2015-04-01

In recent years there has been a call for increased community physical activity and sport programs for female youth that are deliberately structured to foster positive developmental outcomes. In addition, researchers have recognized the need to empirically evaluate such programs to ensure that youth are provided with optimal opportunities to thrive. This study represents a utilization-focused evaluation of Girls Just Wanna Have Fun, a female-only physical activity-based life skills community program. A utilization-focused evaluation is particularly important when the evaluation is to help stakeholders utilize the findings in practice. The purpose of this study was twofold: (a) to gain an understanding of the ongoing successes and challenges after year two of program implementation and (b) to examine how the adaptations made based on feedback from the first year evaluation were perceived as impacting the program. From interviews with youth participants and program leaders, three main themes with eight sub-themes emerged. The main themes were: (a) applying lessons learned can make a significant difference, (b) continually implementing successful strategies, and (c) ongoing challenges. Overall, this evaluation represents an important step in understanding how to improve program delivery to better meet the needs of the participants in community-based programming. Copyright © 2014 Elsevier Ltd. All rights reserved.

A reliability program for emergency diesel generators at nuclear power plants: Program structure

International Nuclear Information System (INIS)

Lofgren, E.V.; DeMoss, G.M.; Fragola, J.R.; Appignani, P.L.; Delarche, G.; Boccio, J.

1988-04-01

The purpose of this report is to provide technical guidelines for NRC staff use in the development of positions for evaluating emergency diesel generator (EDG) reliability programs. Such reviews will likely result following resolution of USI A-44 and GSI B-56. The diesel generator reliability program is a management system for achieving and maintaining a selected (or target) level of reliability. This can be achieved by: (1) understanding the factors that control the EDG reliability and (2) then applying reliability and maintenance techniques in the proper proportion to achieve selected performance goals. The concepts and guidelines discussed in this report are concepts and approaches that have been successful in applications where high levels of reliability must be maintained. Both an EDG reliability program process and a set of review items for NRC use are provided. The review items represent a checklist for reviewing EDG reliability programs. They do not, in themselves, constitute a reliability program. Rather, the review items are those distinctive features of a reliability program that must be present for the program to be effective

Sprego Programming

Directory of Open Access Journals (Sweden)

Maria Csernoch

2015-02-01

Full Text Available Spreadsheet management is a border-land between office applications and programming, however, it is rather communicated that spreadsheet is nothing more than an easily handled fun piece. Consequently, the complexity of spreadsheet handling, the unprepared end-users, their problem solving abilities and approaches do not match. To overcome these problems we have developed and introduced Sprego (Spreadsheet Lego. Sprego is a simplified functional programming language in spreadsheet environment, and such as can be used both as introductory language and the language of end-user programmers. The essence of Sprego is that we use as few and simple functions as possible and based on these functions build multilevel formulas. With this approach, similar to high level programming, we are able solve advanced problems, developing algorithmic skills, computational thinking. The advantage of Sprego is the simplicity of the language, when the emphasis is not on the coding but on the problem. Beyond that spreadsheets would provide real life problems with authentic data and tables which students are more interested in than the artificial environment and semi-authentic problems of high level programming languages.

CUMBIN - CUMULATIVE BINOMIAL PROGRAMS

Science.gov (United States)

Bowerman, P. N.

1994-01-01

The cumulative binomial program, CUMBIN, is one of a set of three programs which calculate cumulative binomial probability distributions for arbitrary inputs. The three programs, CUMBIN, NEWTONP (NPO-17556), and CROSSER (NPO-17557), can be used independently of one another. CUMBIN can be used by statisticians and users of statistical procedures, test planners, designers, and numerical analysts. The program has been used for reliability/availability calculations. CUMBIN calculates the probability that a system of n components has at least k operating if the probability that any one operating is p and the components are independent. Equivalently, this is the reliability of a k-out-of-n system having independent components with common reliability p. CUMBIN can evaluate the incomplete beta distribution for two positive integer arguments. CUMBIN can also evaluate the cumulative F distribution and the negative binomial distribution, and can determine the sample size in a test design. CUMBIN is designed to work well with all integer values 0 < k <= n. To run the program, the user simply runs the executable version and inputs the information requested by the program. The program is not designed to weed out incorrect inputs, so the user must take care to make sure the inputs are correct. Once all input has been entered, the program calculates and lists the result. The CUMBIN program is written in C. It was developed on an IBM AT with a numeric co-processor using Microsoft C 5.0. Because the source code is written using standard C structures and functions, it should compile correctly with most C compilers. The program format is interactive. It has been implemented under DOS 3.2 and has a memory requirement of 26K. CUMBIN was developed in 1988.

Workplace Violence Training Programs for Health Care Workers: An Analysis of Program Elements.

Science.gov (United States)

Arbury, Sheila; Hodgson, Michael; Zankowski, Donna; Lipscomb, Jane

2017-06-01

Commercial workplace violence (WPV) prevention training programs differ in their approach to violence prevention and the content they present. This study reviews 12 such programs using criteria developed from training topics in the Occupational Safety and Health Administration's (OSHA) Guidelines for Preventing Workplace Violence for Healthcare and Social Service Workers and a review of the WPV literature. None of the training programs addressed all the review criteria. The most significant gap in content was the lack of attention to facility-specific risk assessment and policies. To fill this gap, health care facilities should supplement purchased training programs with specific training in organizational policies and procedures, emergency action plans, communication, facility risk assessment, and employee post-incident debriefing and monitoring. Critical to success is a dedicated program manager who understands risk assessment, facility clinical operations, and program management and evaluation.

Veterans Choice Program

Data.gov (United States)

Department of Veterans Affairs — If you are already enrolled in VA health care, the Choice Program allows you to receive health care within your community. Using this program does NOT impact your...

Functional Programming Using F#

DEFF Research Database (Denmark)

Hansen, Michael Reichhardt; Rischel, Hans

This comprehensive introduction to the principles of functional programming using F# shows how to apply basic theoretical concepts to produce succinct and elegant programs. It demonstrates the role of functional programming in a wide spectrum of applications including databases and systems....... Coverage also includes advanced features in the .NET library, the imperative features of F# and topics such as text processing, sequences, computation expressions and asynchronous computation. With a broad spectrum of examples and exercises, the book is perfect for courses in functional programming...... and for self-study. Enhancing its use as a text is an accompanying website with downloadable programs, lecture slides, a mini-projects and links to further F# sources....

76 FR 43571 - Small Business HUBZone Program; Government Contracting Programs

Science.gov (United States)

2011-07-21

... SMALL BUSINESS ADMINISTRATION 13 CFR Part 126 RIN 3245-AG45 Small Business HUBZone Program; Government Contracting Programs AGENCY: U.S. Small Business Administration. ACTION: Interim final rule with request for comments. SUMMARY: This interim final rule amends the U.S. Small Business Administration's...

National Toxicology Program

Science.gov (United States)

... NTP? NTP develops and applies tools of modern toxicology and molecular biology to identify substances in the ... depend on for decisions that matter. The National Toxicology Program provides the scientific basis for programs, activities, ...

Mississippi Curriculum Framework for Welding and Cutting Programs (Program CIP: 48.0508--Welder/Welding Technologist). Postsecondary Programs.

Science.gov (United States)

Mississippi Research and Curriculum Unit for Vocational and Technical Education, State College.

This document, which is intended for use by community and junior colleges throughout Mississippi, contains curriculum frameworks for the course sequences in the welding and cutting programs cluster. Presented in the introductory section are a description of the program and suggested course sequence. Section I lists baseline competencies, and…