Internal Reference Gene Selection for Quantitative Real-Time RT-PCR Normalization in Potato Tissues

摘要

Quantitative real-time PCR(qRT-PCR)is widely used for investigating gene expression patterns and has many advantages,including its high sensitivity,fidelity,and specificity.Selecting a satisfactory internal reference gene is crucial for obtaining precise gene expression results in qRT-PCR analyses.In this study,the transcriptomic data of 2 potato varieties were screened for housekeeping genes with stable expression patterns.A total of 77 putative genes were selected,which were highly and stably expressed.Then,qRT-PCR analyses were performed to examine the expression levels of these 77 candidate reference genes in various potato tissues,including leaves,flowers,stolons,and tubers.Gene expression was represented by analyzing the Ct values at given threshold.Through geNorm and NormFinder program analyses,10 candidate genes with the most stable expression patterns were obtained,including RPL19,RPS15,RPS9,EF1α,TrxP1,RPS8,NTF,CAM,AACM,and RPS28.Moreover,through the comprehensive analyses of 4 statistical algorithms(i.e.,geNorm,NormFinder,BestKeeper,and RefFinder),results indicated that the most appropriate internal reference genes were RPL19 and EF1α.The obtained stable reference genes will contribute to future qRT-PCR analyses on potato tissue-related gene expression.