Liu Meichen, Wang Qun, Xie Hongmei, Liu Shichao, Wang Siming, Zhang Hui, Zhao Yu
Center of Chinese Medicine and Bio‑Engineering Research, Changchun University of Chinese Medicine, Changchun, Jilin 130117, P.R. China.
Mol Med Rep. 2017 Jun;15(6):4382-4390. doi: 10.3892/mmr.2017.6494. Epub 2017 Apr 21.
Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) analysis relies on normalization against a consistently expressed reference gene. However, it has been reported that reference gene expression levels often vary markedly between samples as they are usually selected based solely on convention. The advent of RNA sequencing technology offers the opportunity to select reference genes with the least variability in steady‑state transcript levels. To identify the most consistently stable genes, which are a prerequisite for obtaining reliable gene expression data, the present study analyzed transcriptomes from six Panax ginseng transcriptome data sets, representing six growth stages, and selected 21 candidate reference genes for screening using RT‑qPCR. Of the 21 candidate genes, 13 had not been reported previously. The geNorm, NormFinder and BestKeeper programs were used to analyze the stability of the 21 candidate reference genes. The results showed that UDP‑N‑acetylgalactosamine transporter and nuclear transport factor 2 were likely to be the optimal combination of reference genes for use in investigations of ginseng. The novel reference genes were validated by correlating the gene expression profiles of four pathogenesis‑related protein genes generated from RT‑qPCR, with their expression levels calculated from the RNA sequencing data. The expression levels were well correlated, which demonstrated their value in performing RT‑qPCR analyses in ginseng.
逆转录定量聚合酶链反应(RT-qPCR)分析依赖于针对持续表达的内参基因进行标准化。然而,据报道,由于内参基因通常仅基于惯例选择,其表达水平在不同样本间往往差异显著。RNA测序技术的出现为选择稳态转录水平变化最小的内参基因提供了机会。为了鉴定最稳定的基因(这是获得可靠基因表达数据的前提条件),本研究分析了来自代表六个生长阶段的六个人参转录组数据集的转录组,并选择了21个候选内参基因进行RT-qPCR筛选。在这21个候选基因中,有13个此前未曾报道过。使用geNorm、NormFinder和BestKeeper程序分析了这21个候选内参基因的稳定性。结果表明,UDP-N-乙酰半乳糖胺转运蛋白和核转运因子2可能是人参研究中内参基因的最佳组合。通过将RT-qPCR产生的四个病程相关蛋白基因的基因表达谱与其从RNA测序数据计算得到的表达水平进行关联,对新的内参基因进行了验证。表达水平具有良好的相关性,这证明了它们在人参RT-qPCR分析中的价值。