Dataset Information

Selection and Validation of Reference Genes for Gene Expression Analysis in Vigna angularis Using Quantitative Real-Time RT-PCR.

ABSTRACT: Adzuki bean (Vigna angularis) is one of the most important legume crops in Asian countries like China, Japan and Korea due to its nutritious protein and starch contents. In spite of its economic importance, gene expression analysis system for gene function verification of adzuki bean is still absent. Therefore, reference genes for gene expression analysis based on the quantitative real time PCR (qRT-PCR) were screened in current study. A total of nine general housekeeping genes, including ACT, Fbox, ZMPP, GAPDH, EF, PP2A, UBC, UBN and PTB were evaluated for their expression stability by qRT-PCR in four adzuki bean cultivars, three different tissues, four abiotic stress and one biotic stress. The best group of candidates as reference genes were as follows: PTB and ACT for different cultivars; EF and UBN for different tissues; ACT and ZMPP for biotic stress and waterlogging stress; Fbox and UBC for salinity-alkalinity stress; Fbox and PTB for drought stress. Our results will provide a more accurate and reliable normalization of qRT-PCR data in adzuki bean.

SUBMITTER: Chi C

PROVIDER: S-EPMC5161372 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Selection and Validation of Reference Genes for Gene Expression Analysis in Vigna angularis Using Quantitative Real-Time RT-PCR.

Chi Chao C Shen Yongqiang Y Yin Lihua L Ke Xiwang X Han Dong D Zuo Yuhu Y

PloS one 20161216 12

Adzuki bean (Vigna angularis) is one of the most important legume crops in Asian countries like China, Japan and Korea due to its nutritious protein and starch contents. In spite of its economic importance, gene expression analysis system for gene function verification of adzuki bean is still absent. Therefore, reference genes for gene expression analysis based on the quantitative real time PCR (qRT-PCR) were screened in current study. A total of nine general housekeeping genes, including ACT, F ...[more]

PMID: 27992593

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Project description:BackgroundWith the availability of genome sequences, gene expression analysis of jute has drawn considerable attention for understanding the regulatory mechanisms of fiber development and improving fiber quality. Gene expression profiles of a target gene can provide valuable clues towards the understanding of its biological function. Reverse transcription quantitative real-time PCR (qRT-PCR) is the best method for targeted gene expression analysis due to its sensitivity and reproducibility. However, calculating relative expression requires reference genes, which must be stable across various biological conditions. For this purposes, 11 prospective genes namely, 28S RNA, ACT7, CYP, EF1A, EF2, ETIF3E, GAPDH, PP2Ac, PTB, UBC2 and UBI1 were evaluated for their potential use as reference genes in jute.ResultsThe expression stabilities of eleven prospective genes were analyzed in various jute plant tissues, such as the root, stick, bark, leaf, flower, seed and fiber, as well as under abiotic (waterlogged, drought and salinity) and biotic stress (infestation with Macrophomina phaseolina) conditions with different time points. All 11 genes were variably expressed in different tissues and stress conditions. To find suitable reference genes in different sample sets, a comprehensive approach based on four statistical algorithms such as GeNorm, BestKeeper, NormFinder the ?Ct was used. The PP2Ac and EF2 genes were the most stably expressed across the different tissues. ACT7 and UBC2 were suitable reference genes under drought stress, and CYP and PP2Ac were the most appropriate after inoculation with Macrophomina phaseolina. Under salinity stress, PP2Ac and UBC2 were the best genes, and ACT7 and PP2Ac were the most suitable under waterlogged conditions.ConclusionExpression stability of reference genes from jute varied in different tissues and selected experimental conditions. Our results provide a valuable resource for the accurate normalization of gene expression experiments in fiber research for important bast fiber crops.

Project description:BackgroundReal-time RT-PCR is the recommended method for quantitative gene expression analysis. A compulsory step is the selection of good reference genes for normalization. A few genes often referred to as HouseKeeping Genes (HSK), such as ACT1, RDN18 or PDA1 are among the most commonly used, as their expression is assumed to remain unchanged over a wide range of conditions. Since this assumption is very unlikely, a geometric averaging of multiple, carefully selected internal control genes is now strongly recommended for normalization to avoid this problem of expression variation of single reference genes. The aim of this work was to search for a set of reference genes for reliable gene expression analysis in Saccharomyces cerevisiae.ResultsFrom public microarray datasets, we selected potential reference genes whose expression remained apparently invariable during long-term growth on glucose. Using the algorithm geNorm, ALG9, TAF10, TFC1 and UBC6 turned out to be genes whose expression remained stable, independent of the growth conditions and the strain backgrounds tested in this study. We then showed that the geometric averaging of any subset of three genes among the six most stable genes resulted in very similar normalized data, which contrasted with inconsistent results among various biological samples when the normalization was performed with ACT1. Normalization with multiple selected genes was therefore applied to transcriptional analysis of genes involved in glycogen metabolism. We determined an induction ratio of 100-fold for GPH1 and 20-fold for GSY2 between the exponential phase and the diauxic shift on glucose. There was no induction of these two genes at this transition phase on galactose, although in both cases, the kinetics of glycogen accumulation was similar. In contrast, SGA1 expression was independent of the carbon source and increased by 3-fold in stationary phase.ConclusionIn this work, we provided a set of genes that are suitable reference genes for quantitative gene expression analysis by real-time RT-PCR in yeast biological samples covering a large panel of physiological states. In contrast, we invalidated and discourage the use of ACT1 as well as other commonly used reference genes (PDA1, TDH3, RDN18, etc) as internal controls for quantitative gene expression analysis in yeast.

Project description: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 or cells under investigation. Quantitative assessment and control of qRT-PCR inhibitors using an external RNA control can reduce the variation of qRT-PCR assay and facilitate the evaluation of gene stability. Our results may facilitate the choice of reference genes for expression studies in HCC.

Dataset Information

Selection and Validation of Reference Genes for Gene Expression Analysis in Vigna angularis Using Quantitative Real-Time RT-PCR.

Publications

Selection and Validation of Reference Genes for Gene Expression Analysis in Vigna angularis Using Quantitative Real-Time RT-PCR.

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