Transcriptome-based screening and the optimal reference genes for real-time quantitative PCR in Rehmannia chingii and R. henryi

Abstract

Real time quantitative PCR (qPCR) is a powerful tool for studying the expression of specific genes. The accuracy and reliability of qPCR analysis data require the selection of reference genes with stable expression. However, the reference genes that can be used for qPCR of Rehmannia chingii and R. henryi have not yet been identified. In this study, based on the transcriptome data of R. chingii and R. henryi, we initially selected genes with relatively stable expression in different samples. We screened six candidate reference genes in R. chingii and R. henryi and calculated their expression abundance by real time qPCR. Their expression stability was evaluated by three algorithms geNorm, NormFinder, and BestKeeper. Although the results obtained by different algorithms were not completely consistent, R. chingii type 2A phosphatase activator TIP41 and R. chingii 18S ribosomal RNA had the highest expression stability in six different samples of R. chingii, and R. henryi 18S ribosomal RNA and R. henryi actin showed the most stable expression in different samples of R. henryi. In addition, based on transcriptome data, four genes were screened in R. chingii and R. henryi, and the expression stability of the selected reference genes was further verified. This study laid the foundation for further analysis and verification of the functions of important genes in R. chingii and R. henryi. Additional key words: BestKeeper, gene expression stability, geNorm, NormFinder. Submitted 13 July 2020, last revision 18 October 2020, accepted 19 October 2020. Abbreviations: 4CL 4-Coumarate-CoA ligase; C4H cinnamic acid 4-hydroxylase; Cq quantitative cycle; CV coefficient of variation; EF-1α elongation factor 1-alpha; ERF ethylene-responsive transcription factor; FPKM fragments per kilobase of transcript per million fragments mapped; GAPDH glyceraldehyde 3-phosphate dehydrogenase; M value gene expression stability measure; ORF open reading frame; PAL phenylalanine ammonia-lyase; 18S 18S ribosomal RNA; Tm melting temperature; UBQ ubiquitin. Acknowledgements. This research was supported by the National Natural Science Foundation of China (Grant No. 81872950) and the National Key Research and Development Program (2017YFC1700705). * Corresponding author; e-mail: heauzycxw@126.com This is an open access article distributed under the terms of the Creative Commons BY-NC-ND Licence