小鼠卵巢组织定量PCR分析中内参基因的筛选

doi:10.11843/j.issn.0366-6964.2019.02.024

摘要/Abstract

摘要：

旨在选择小鼠卵巢组织中合适的内参基因，为研究卵巢发育过程中基因表达提供可靠数据。以不同年龄（0日龄、3周龄、5周龄、8周龄）小鼠卵巢组织为试验材料，Trizol法提取样本总RNA，并合成cDNA，根据已报道的小鼠（Mus musculus）各组织中相对稳定表达的8个常见内参基因（Gapdh、β-actin、β-tubulin、18S rRNA、16S rRNA、H2afz、Ubc、Rpl13a）的GenBank登录序列设计引物，采用实时荧光定量PCR（qRT-PCR）方法构建卵巢cDNA等梯度（1：10）稀释后的标准曲线；利用geNorm分析候选内参基因的表达稳定性（M）；NormFinder筛选表达最稳定的内参基因；BestKeeper计算qRT-PCR结果的标准差（SD）和变异系数（CV），从而预测候选内参基因的稳定性。结果表明，8个内参基因的引物特异性较强，具有良好的线性关系；候选内参基因的表达稳定度排序：Gapdh=β-actin > 18S rRNA > Ubc > 16S rRNA > H2afz > Rpl13a > β-tubulin；其中 Gapdh表达稳定性最好，且标准差及方差系数最小（SD：0.32，CV：1.95），H2afz标准差及方差系数最大（SD>1.0，CV：5.76）。综上表明，本研究成功获得出生后小鼠卵巢发育过程中稳定表达的内参基因（Gapdh和β-actin），可作为其基因表达研究中的最佳候选内参。

Abstract:

The aim of this study was to screen the suitable reference gene in mouse ovaries, which provided important foundation for mRNA expression analysis during ovary development. Mouse ovaries tissues at 4 different development stages (0 day, 3 weeks, 5 weeks, 8 weeks old) were selected as experimental materials. The Trizol method was used to extract the total RNA and synthesize cDNA. The primers of 8 commonly used internal reference genes (Gapdh, β-actin, β-tubulin, 18S rRNA, 16S rRNA, H2afz, Ubc, Rpl13a) were designed based on the sequence of Mus musculus. Ovaries cDNA were diluted gradiently (1:10) to construct standard curves by qRT-PCR. GeNorm was used to calculate gene expression stability measure (M) of the 8 candidated reference genes. NormFinder analysis was performed to compare the stability between selected reference genes, and BestKeeper ranked the standard deviation (SD) and coefficient of variance (CV) of candidate gene expression to determine the optimal reference gene. The result indicated that the primers of 8 internal reference genes were highly specific and had good linear relationship. According to the results of geNorm, the stabilities of selected reference genes were Gapdh=β-actin > 18S rRNA > Ubc > 16S rRNA > H2afz > Rpl13a > β-tubulin. Based on the results of NormFinder, Gapdh had the best stability in the ovaries of mouse as the standard deviation and coefficient of variation were the smallest (SD:0.32, CV:1.95), while H2afz was considered as unstable with SD>1.0, CV=5.76. In conclusion, we have successfully identified Gapdh, β-actin as the most suitable reference genes during ovary development after birth, which could be used as reference genes for normalizing genes analysis.

中图分类号:

S814

杨显英, 熊显荣, 韩杰, 黄向月, 王艳, 阿果约达, 李键. 小鼠卵巢组织定量PCR分析中内参基因的筛选[J]. 畜牧兽医学报, 2019, 50(2): 446-453.

YANG Xianying, XIONG Xianrong, HAN Jie, HUANG Xiangyue, WANG Yan, A GUO Yueda, LI Jian. Identification of Suitable Reference Gene for Quantitative RT-PCR Analysis in Mouse Ovaries[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(2): 446-453.

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