干扰MSTN的牛骨骼肌卫星细胞实时荧光定量PCR内参基因的筛选

doi:10.16431/j.cnki.1671-7236.2020.02.008

Abstract

Abstract: In order to screen the reference genes that suitable for bovine skeletal muscle satellite cells (MSCs) differentiation process and not affected by MSTN gene expression,bovine MSCs from wild group (WT),MSTN interfered group (si-MSTN) and control group (NC-MSTN) were used as sample.HMBS,B2M,GAPDH,TUBB,SDHA,18S rRNA,ACTB,RPL4,PPIA,HPRT1 and YWHAZ were selected as candidate internal reference genes.The relative expression of candidate internal reference genes was determined by Real-time quantitative PCR and Ct value analysis.Firstly,the expression stability of each candidate internal reference gene in wild group (WT) bovine MSCs of proliferative (GM) and the differentiated third day after differention (DM3) cells was evaluated by three independent evaluation softwares:geNorm,NormFinder and BestKeeper,and the first five candidate reference genes with the most stable expression were screened out.Furthermore,the expression level and stability of these five internal reference genes in si-MSTN group and NC-MSTN group of GM and DM3 were analyzed by similar methods.The analysis of geNorm and NormFinder showed that the expression of HMBS,B2M,TUBB,GAPDH and ACTB genes were more stable before and after differentiation of bovine MSCs.The BestKeeper showed that the correlation coefficient (R) of ACTB and TUBB were ranked first,GAPDH,HMBS and B2M were not very high,but GAPDH had the lowest SD value,so these five candidate genes were selected for subsequent experiments.In siRNA-MSTN group and NC-MSTN group of GM and DM3,the results of geNorm and NormFinder showed that the expressions of GAPDH,TUBB and B2M in the five candidate reference genes were more stable than other.BestKeeper showed that the correlation coefficient of TUBB was not the highest,but its SD value was the lowest.Based on the above analysis,TUBB was selected as the most suitable internal reference gene in GM and DM3 of bovine MSCs siRNA-MSTN and NC-MSTN groups.The result of this study would provide reference for future research on gene expression analysis of bovine MSCs in different growth process and after MSTN expression was regulated.

Key words: MSTN; bovine; MSCs; internal reference genes; stability

CLC Number:

S813.3

SHENG Hui, ZHANG Linlin, GUO Yiwen, LI Xin, DING Xiangbin, GUO Hong. A Selection of Reference Genes for Real-Time Quantitative PCR in Bovine Skeletal Muscle Satellite Cells Interfering with MSTN[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(2): 381-391.

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A Selection of Reference Genes for Real-Time Quantitative PCR in Bovine Skeletal Muscle Satellite Cells Interfering with MSTN

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