Identification of optimal endogenous reference RNAs for RT-qPCR normalization in hindgut of rat models with anorectal malformations
Identification of optimal endogenous reference RNAs for RT-qPCR normalization in hindgut of rat models with anorectal malformations
复制标题
鉴定用于肛门直肠畸形大鼠模型后肠 RT-qPCR 标准化的最佳内源参考 RNA
作者：
Long Caiyun;Xiao Yunxia;Li Siying;Tang Xiaobing;Yuan Zhengwei;Bai Yuzuo
实时定量聚合酶链反应（RT-qPCR）是一种灵敏的定量mRNA丰度的方法。然而，对于相对表达分析，可靠的数据输出依赖于在所研究的样品中稳定表达的参考基因。在肛门直肠畸形（ARM），有有限的数据选择适当的参考基因。目的探讨ARM大鼠模型PCR扩增的最佳参考基因。方法选取15个常用参考基因（Rps 18、Actb、B2 m、Gapdh、Ppia、Hprt 1、Pgk 1、Ywhaz、Tbp、Ubc、Rps 16、Rpl 13 a、Rplp 1、Sdha和Hmbs）作为候选参考基因，采用RT-qPCR方法检测其在ARM标本中的mRNA表达。随后使用四种方法（geNorm、Normalization、比较ΔCt和BestKeeper）评价这些转录物的表达稳定性和变异性。结果RT-qPCR检测结果表明候选参考基因的丰度符合要求，循环阈值（Ct）在14.07（Rplp 1）~ 21.89（Sdha）之间。在整个候选基因中，不同的算法存在不同的变异。综合分析表明，Rpl 13 a在相对稳定的基因中排名第一，其次是Ywhaz，Rps 18，Sdha和Hmbs。结论RT-qPCR检测ETU诱导的大鼠胚胎ARMs中最稳定的参考基因为Rpl 13 a、Ywhaz和Rps 18。本研究为今后基因表达分析中参考基因的选择奠定了基础。
Background Quantitative real-time polymerase chain reaction (RT-qPCR) is a sensitive method for quantifying mRNA abundance. With relative expression analysis, however, reliable data output is dependent on stably expressed reference genes across the samples being studied. In anorectal malformations (ARMs), there is limited data on the selection of appropriate reference genes. Purpose This study was aimed to investigate the optimal reference genes for PCR in ARM rat models. Methods We selected 15 commonly used reference genes (Rps18, Actb, B2m, Gapdh, Ppia, Hprt1, Pgk1, Ywhaz, Tbp, Ubc, Rps16, Rpl13a, Rplp1, Sdha, and Hmbs) as candidate reference genes and detected their mRNA expression in ARM samples by RT-qPCR. The expression stability and variability of these transcripts were subsequently evaluated using four methods (geNorm, NormFinder, comparative ΔCt, and BestKeeper). Results The abundance of the candidate reference genes was qualified by RT-qPCR and the cycle threshold (Ct) values ranged between 14.07 (Rplp1) and 21.89 (Sdha). In the overall candidate genes, different variations existed across the different algorithms. A comprehensive analysis revealed that Rpl13a ranked first among the relatively stable genes, followed by Ywhaz, Rps18, Sdha, and Hmbs. Conclusions The most stable reference genes for RT-qPCR were Rpl13a, Ywhaz, and Rps18 in ETU-induced ARMs in rat fetus. This study provided a foundation for reference gene selection for future gene expression analyses.
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