Selection and validation of reference genes for RT-qPCR analysis in Merr.

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Gene expression valuated by reverse transcription-quantitative PCR (RT-qPCR) are often applied to study the gene function. To obtain accurate and reliable results, the usage of stable reference genes is essential for RT-qPCR analysis. The traditional southern Chinese medicinal herb, Merr is well known for its remarkable effect on the treatment of urination disturbance, urolithiasis, edema and jaundice. However, there are no ready-made reference genes identified for . In this study, 13 novel genes retrieved from transcriptome datasets of four different tissues were reported according to the coefficient of variation (CV) and maximum fold change (MFC) of gene expression. The expression stability of currently used Leguminosae was compared to the 13 candidate reference genes in different tissues and 7-day-old seedlings under different experimental conditions, which was evaluated by five statistical algorithms (geNorm/NormFinder/BestKeeper/ΔCT/RefFinder). Our results indicated that the reference gene combinations of  +  ,  +  and  +  were the most stable reference genes in leaf, stem and root tissues, respectively. The most stable reference gene combination for all tissues was  +  . In addition, the most stable reference genes for different experimental conditions were distinct, for instance for MeJA treatment,  +  for SA treatment,  +   for ABA treatment and  +  for salt stress, respectively. Our results lay a foundation for achieving accurate and reliable RT-qPCR results so as to correctly understand the function of genes in .

SUPPLEMENTARY INFORMATION

The online version contains supplementary material available at 10.1007/s13205-021-02954-x.