Selection and validation of reference genes for quantitative real-time PCR in Cymbidium sinense

Abstract

Selection of reference genes (RGs) is important for the accurate analysis of real-time quantitative PCR (RT-qPCR) results. This study screened RGs of Cymbidium sinense for more accurate quantification of target genes. The two most stable RGs for all tissues were ACT and EF1α, those for vegetative organs were UBQ3 and ACT, while those for reproductive organs as well as organs in the full flowering stage were EF1α and ACT. The AGAMOUS (CsAG1) expression level was verified using EF1α, ACT, GAPDH, UBQ2 and UBQ3 as RG. The expression profile of CsAG1 was consistent when normalized with EF1α, ACT and UBQ3. The results have practical value for the expression of key genes during the development of C. sinense. METHOD SUMMARY To screen reference genes (RGs) in Cymbidium sinense for more accurate quantification of target genes, the expression levels of candidate RGs were detected by RT-qPCR. The expression stability of these six candidate RGs was evaluated using geNorm, NormFinder, BestKeeper, DeltaCt and the comprehensive evaluation software RefFinder. To further verify the stability of the six candidate RGs, the expression of target gene AG was detected.