Identification and validation of reference genes for RT-qPCR analysis in fetal rat pancreas
Identification and validation of reference genes for RT-qPCR analysis in fetal rat pancreas
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胎鼠胰腺 RT-qPCR 分析参考基因的鉴定和验证
DOI：
10.1016/j.reprotox.2021.09.009
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发表时间：
2021-09-20
影响因子：
3.3
通讯作者：
Guo, Yu
中科院分区：
文献类型：
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作者：
Dai, Yongguo;Kou, Hao;Guo, Yu
参考基因的选择是定量逆转录-聚合酶链反应（RTqPCR）检测的关键。为了筛选和确定合适的胎鼠胰腺参考基因，我们选择了8个候选参考基因（Gapdh，Actb，Rn 18 s，B2 m，Rp 113 a，Tbp，Ywhaz和Ubc），采用GeNorm，Normandise，Bestkeeper和Comparative Delta Ct方法，对胎鼠胰腺在非病理状态和产前地塞米松暴露（PDE）模型中基因表达的恒定性进行了评价。此外，比较对照组和PDE组之间胰腺胰岛素mRNA水平的变化，以验证所选参考基因用于RT-qPCR数据标准化的可靠性。生理条件下参考基因的综合排序为：Gapdh > Actb > Ywhaz > Ubc > Rn 18 s> Rpl 13 a> B2 m> Tbp（雌性）; Actb > Ywhaz > Gapdh > Ubc > B2 m> Rpl 13 a> Rn 18 s| Tbp（男性）。排名靠前的参考基因在PDE胎儿胰腺中也稳定表达。最佳参考基因组合分别为：雌性胎鼠胰腺的Ywhaz+Actb和雄性胎鼠胰腺的Ywhaz+Gapdh。对照组和PDE组胰岛素mRNA的最佳参考基因组合标准化后的变化趋势较低等级或单一参考基因更显著，且与血清胰岛素水平变化趋势一致。本研究结果为RT-qPCR检测胰腺发育毒性提供了最佳的参考基因组合。
The choice of reference gene is crucial for quantitative reverse transcriptase-polymerase chain reaction (RTqPCR) assay. To screen and determine the suitable reference genes in fetal rat pancreas, we selected eight candidate reference genes (Gapdh, Actb, Rn18 s, B2m, Rpl13a, Tbp, Ywhaz and Ubc), and evaluated the constancy of gene expression from fetal rat pancreases in non-pathological situation and prenatal dexamethasone exposure (PDE) model, using four algorithms: GeNorm, NormFinder, Bestkeeper and Comparative Delta Ct method. In addition, the alteration of mRNA levels of pancreatic insulin was compared between control and PDE groups to validate the reliability of selected reference genes for data normalization of RT-qPCR. The comprehensive ranking of reference genes under physiological condition was as follow: Gapdh > Actb > Ywhaz > Ubc > Rn18s > Rpl13a > B2m > Tbp (female); Actb > Ywhaz > Gapdh > Ubc > B2m > Rpl13a > Rn18 s | Tbp (male). The top ranking reference genes were also stably expressed in PDE fetal pancreas. The best reference gene combinations are: Ywhaz+Actb for female and Ywhaz+Gapdh for male fetal rat pancreas, respectively. Compared with low ranking or single reference gene, the change trend of insulin mRNA normalized by the best reference gene combination between control and PDE groups was more significant and consistent with that of serum insulin level. In conclusion, our results provided the optimal combination of stable reference genes for RT-qPCR assay in pancreatic developmental toxicity study.