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

Abstract

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.

CLC Number:

S814

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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Identification of Suitable Reference Gene for Quantitative RT-PCR Analysis in Mouse Ovaries

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