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实时定量PCR(qPCR)技术广泛应用于基因表达量的检测,为了保证其结果的准确性,选择合适的内参基因是必不可少的环节。本研究分析了11个玉米候选内参基因的稳定性,其中7个是常用内参基因(GAPDH、ACT、EF-1α、γ-TUB、18S rRNA、5S rRNA和U6 snRNA),4个是microRNA(zma-miR171a、zma-miR171b、zma-miR172和zma-miR159a/b)。通过计算11个基因在正常生长条件与干旱、盐和碱共胁迫条件下的循环阈值(Ct),输入软件geNorm、Norm Finder和Best Keeper中进行分析。结果表明,microRNA比其他候选内参基因稳定,其中zma-miR172和zma-miR171a最稳定,18S rRNA最不稳定。另外,选择zma-miR397a及其靶基因LAC4对候选内参基因进行验证,发现不合适的内参基因可能会导致错误的结果。
Real-time quantitative PCR (qPCR) technology is widely used in the detection of gene expression, in order to ensure the accuracy of the results, select the appropriate internal control gene is an essential part. In this study, we analyzed the stability of 11 maize candidate reference genes, of which 7 are commonly used reference genes (GAPDH, ACT, EF-1α, γ-TUB, 18S rRNA, 5S rRNA and U6 snRNA) -miR171a, zma-miR171b, zma-miR172 and zma-miR159a / b). The cycle threshold (Ct) of 11 genes under normal growth conditions and drought, salt and alkali co-stress conditions was calculated and input into the software geNorm, Norm Finder and Best Keeper for analysis. The results showed that the microRNAs were more stable than other candidate reference genes, of which zma-miR172 and zma-miR171a were the most stable and the 18S rRNA was the most unstable. In addition, zma-miR397a and its target gene LAC4 were selected to validate the candidate internal reference genes, and found that inappropriate internal reference genes may lead to wrong results.