Internal Reference Gene Selection For Quantitative Real-Time RT-PCR Normalization In Potato
Quantitative real-time RT-PCR (qRT-PCR), which is widely used for investigating gene expression patterns, has many advantages such as high sensitivity, high fidelity, and high specificity. Selecting a satisfactory internal reference gene is crucial to precise result of gene expression in qRT-PCR experiments. In the present study, the transcriptomic data of two potato varieties were used to screen housekeeping genes with stable expression patterns. A total of 77 putative genes were chose which were highly and stably expressed. Then qRT-PCR experiments were performed to examine the expression levels of these 77 candidate reference genes in potato tissues including leaves, flowers, stolons, and tubers. The gene expression was represented by analyzing the Ct values at given threshold. Through geNorm and NormFinder program analyses, ten candidate genes with most stable expression patterns were got, including RPL19, RPS15, RPS9, EF1α, TrxP1, RPS28, NTF, CAM, AACM, and RPS28. Moreover, through comprehensive analyses of four statistical algorithms geNorm, NormFinder, BestKeeper and RefFinder, the results indicated that the most appropriate internal reference genes were RPL19 and EF1α. The obtained identifications of stable reference genes will contribute to subsequent qRT-PCR analysis of gene expression related to potato tissues.
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