Abstract
Real-time quantitative polymerase chain reaction (RT-qPCR) has been widely applied in gene expression and transcription abundance analysis because of its high sensitivity, good repeatability, and strong specificity. Selection of relatively stable reference genes is a precondition in order to obtain the reliable analysis results. However, little is known about evaluation of a set of reference genes through scientific experiments in Rubia plants. Here, 15 candidate reference genes were selected from R. yunnanensis transcriptome database and analyzed under abiotic stresses, hormone treatments, and different tissues. Among these 15 candidate reference genes, heterogeneous nuclear ribonucleoprotein (hnRNP), TATA binding protein (TBP), ribosomal protein L5 (RPL5), malate dehydrogenase (MDH), and elongation factor 1-alpha (EF-1α) were indicated as the five most stable reference genes by four statistical programs (geNorm, NormFinder, BestKeeper, and RefFinder). Ultimately, the validity of reference genes was confirmed by normalizing the expression of o-succinylbenzoate-CoA ligase (OSBL) and isochorismate synthase (ICS) involved in the anthraquinone biosynthesis pathway in different tissues and hormone treatments. Meanwhile, four other putative genes involved in the anthraquinone biosynthesis pathway were also normalized with the selected reference genes, which showed similar expression levels with those given by transcriptome data. This work is the first research that aims at a systematic validation on the stability of reference genes selected from R. yunnanensis transcriptome data and will be conducive to analyze gene expression in R. yunnanensis or other Rubia species.
Highlights
Real-time quantitative polymerase chain reaction (RTqPCR) is a technique for precise quantification of nucleic acids by monitoring the entire PCR process using a real-time fluorescence quantifier
Reference genes are usually housekeeping genes, which are expressed in all kinds of cells in organisms, and their products are essential proteins to maintain the basic life activities of cells. ere are hundreds of housekeeping genes, among which GAPDH, ACT, TUB, 18S rRNA, and 28S rRNA are often used as internal reference genes for the standardization of target genes owing to their stable expression levels under different conditions and various tissues in many plant
A single amplification peak for each candidate reference gene was observed in the melting curve (Figure 2). e PCR amplification efficiencies for these genes ranged from 93.36% for PTBP2 to 108.08% for UBCE, and the correlation coefficients varied from 0.999 to 0.990 (Table 1)
Summary
Real-time quantitative polymerase chain reaction (RTqPCR) is a technique for precise quantification of nucleic acids by monitoring the entire PCR process using a real-time fluorescence quantifier It plays an extremely important role in gene expression analysis and is the most extensive method compared with the other three methods (northern blot, microarray, and high-throughput sequencing) due to its high sensitivity, good repeatability, strong specificity, high throughput, wide application, and low cost [1]. Erefore, it is completely necessary to select suitable internal reference genes as the standard to study the expression levels of target genes according to the different sample types and test conditions in order to make the results more reliable. Many novel reference genes were validated, and their suitability was evaluated by employing these programs in different plants, such as Euscaphis konishii [11], Sapium sebiferum [12], and Neolamarckia cadamba [13]
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