Abstract
BackgroundQuantitative PCR (qPCR) is one of the most common and accurate methods of gene expression analysis. However, the biggest challenge for this kind of examinations is normalization of the results, which requires the application of dependable internal controls. The selection of appropriate reference genes (RGs) is one of the most crucial points in qPCR data analysis and for correct assessment of gene expression. Because of the fact that many reports indicate that the expression profiles of typically used RGs can be unstable in certain experimental conditions, species or tissues, reference genes with stable expression levels should be selected individually for each experiment. In this study, we analysed a set of ten candidate RGs for wheat seedlings under short-term drought stress. Our tests included five ‘traditional’ RGs (GAPDH, ACT, UBI, TUB, and TEF1) and five novel genes developed by the RefGenes tool from the Genevestigator database.ResultsExpression stability was assessed using five different algorithms: geNorm, NormFinder, BestKeeper, RefFinder and the delta Ct method. In the final ranking, we identified three genes: CJ705892, ACT, and UBI, as the best candidates for housekeeping genes. However, our data indicated a slight variation between the different algorithms that were used. We revealed that the novel gene CJ705892, obtained by means of in silico analysis, showed the most stable expression in the experimental tissue and condition.ConclusionsOur results support the statement, that novel genes selected for certain experimental conditions have a more stable level of expression in comparison to routinely applied RGs, like genes encoding actin, tubulin or GAPDH. Selected CJ705892 gene can be used as a housekeeping gene in the expression analysis in wheat seedlings under short-term drought. The results of our study will be useful for subsequent analyses of gene expression in wheat tissues subjected to drought.
Highlights
Quantitative PCR is one of the most common and accurate methods of gene expression analysis
Selection of candidate reference genes using the RefGenes tool RefGenes is an in silico method enabling the identification of genes with high expression stability within microarray libraries of wheat subjected to drought
Our results indicated that a novel gene obtained using the RefGenes tool from the Genevestigator database was the most stable among all the tested genes
Summary
Quantitative PCR (qPCR) is one of the most common and accurate methods of gene expression analysis. The selection of appropriate RGs is one of the most crucial points in qPCR data analysis and for correct assessment of gene expression [4, 5] Numerous housekeeping genes, such as actin (ACT), tubulin (TUB), and 18S ribosomal RNA (18S rRNA), that are necessary for proper cellular metabolism are widely used as RGs in many studies. The Genevestigator database contains a large set of systematically annotated and quality-controlled microarray data from several organisms [22], and RefGenes is an online tool that utilizes this database to enable users to search for genes that exhibit minimal expression variance across a chosen set of arrays This method ensures the identification of genes with more stable expression than the standard genes [1, 2, 16, 23, 24]. For the analysis of the results and selection of the best RG, numerous platforms using different algorithms have been developed, including geNorm [25], NormFinder [26], BestKeeper [27] and RefFinder [28]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
