民猪外周血单核细胞实时荧光定量PCR内参基因的筛选

doi:10.16431/j.cnki.1671-7236.2017.12.005

《中国畜牧兽医》 ›› 2017, Vol. 44 ›› Issue (12): 3426-3433.doi: 10.16431/j.cnki.1671-7236.2017.12.005

民猪外周血单核细胞实时荧光定量PCR内参基因的筛选

马红, 刘宇, 汪亮, 王文涛, 吴赛辉, 何鑫淼, 刘娣

黑龙江省农业科学院畜牧研究所, 哈尔滨 150086

收稿日期:2017-06-23 出版日期:2017-12-20 发布日期:2017-12-20
通讯作者: 刘娣 E-mail:liudi1963@163.com
作者简介:马红(1974-),女,黑龙江虎林人,博士,研究方向:动物遗传育种与繁殖,E-mail:mahong_ok@163.com
基金资助:
黑龙江省科研机构创新能力提升专项计划（YC2016D001）

Screening of the Reference Genes for the Study of Peripheral Blood Mononuclear Cells by Quantitative Real-time PCR in Min Pigs

MA Hong, LIU Yu, WANG Liang, WANG Wen-tao, WU Sai-hui, HE Xin-miao, LIU Di

Institute of Animal Husbandry of Heilongjiang Academy of Agricultural Sciences, Harbin 150086, China

Received:2017-06-23 Online:2017-12-20 Published:2017-12-20

摘要/Abstract

摘要：

民猪是能够适应东北地区寒冷环境的地方猪种，但目前对其环境适应性相关基因的研究较少，也没有确定适合此研究所需的实时荧光定量PCR的内参基因。因此，本试验通过研究常用的12个内参基因（B2M、ACTB、RPL11、RPL4、YWHAZ、GAPDH、HPRT1、SDHA、HMBS、IDH3B、TUBB2B和TBP1）在不同环境温度下民猪外周血单核细胞中的表达稳定性，旨在确定合适的内参基因进行相关研究。分别在-25、5、10和30℃采集3头民猪耳静脉血分离出单核细胞，利用geNorm、NormFinder、BestKeeper 3种软件分析12个候选内参基因的Ct值，筛选出表达稳定的基因作为内参基因。经geNorm和NormFinder计算获得各候选基因的M值发现，12个候选基因的表达均相对稳定，而BestKeeper的分析则显示ACTB、GAPDH、SDHA、HPRT1、TBP1、YWHAZ基因的SD值均<1，可用作本研究条件下的内参基因，而另外6个基因SD值则>1，不符合作为内参基因的标准。综合3种分析的结果，在本研究条件下，TBP1基因的稳定性最高，ACTB、GAPDH、SDHA、HPRT1和YWHAZ基因也符合作为内参基因的标准，都可研究在不同环境温度下民猪外周血单核细胞中基因表达时作为内参基因。

关键词: 民猪; 外周血单核细胞; 环境温度; 实时荧光定量PCR; 内参基因

Abstract:

Min pig is a local pig breed in Northeast China. It has been well-adapted to the local cold weather,but few genes related to its environmental adaptation have been studied. For studies about environmental adaptation of Min pig on molecular level,it is important to have proper reference genes for quantification of gene expression by quantitative Real-time PCR. In this study,12 reference genes (B2M,ACTB,RPL11,RPL4,YWHAZ,GAPDH,HPRT1,SDHA,HMBS,IDH3B,TUBB2B and TBP1) were evaluated for their potential as the reference gene in Min pig peripheral blood mononuclear cells under different temperatures. Blood samples were collected from 3 Min pigs which were under -25,5,10 and 30℃,respectively. Mononuclear cells were separated using density gradient centrifugation. Statistical algorithms including geNorm,Normfinder and BestKeeper were employed to assess the stabilities of these genes. Analysis of geNorm and Normfinder revealed that all these 12 genes were highly stable. However,ACTB,GAPDH,SDHA,HPRT1,TBP1 and YWHAZ genes (SD<1) were found to be more stable than other six genes (SD>1),of which TBP1 was the most stable one using BestKeeper program. To summarize,ACTB,GAPDH,SDHA,HPRT1, TBP1 and YWHAZ genes were suitable to be the reference genes,with TBP1 was the best one.

Key words: Min pig; peripheral blood mononuclear cells; ambient temperature; quantitative Real-time PCR; reference gene

中图分类号:

Q781

马红, 刘宇, 汪亮, 王文涛, 吴赛辉, 何鑫淼, 刘娣. 民猪外周血单核细胞实时荧光定量PCR内参基因的筛选[J]. 《中国畜牧兽医》, 2017, 44(12): 3426-3433.

MA Hong, LIU Yu, WANG Liang, WANG Wen-tao, WU Sai-hui, HE Xin-miao, LIU Di. Screening of the Reference Genes for the Study of Peripheral Blood Mononuclear Cells by Quantitative Real-time PCR in Min Pigs[J]. , 2017, 44(12): 3426-3433.

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民猪外周血单核细胞实时荧光定量PCR内参基因的筛选

Screening of the Reference Genes for the Study of Peripheral Blood Mononuclear Cells by Quantitative Real-time PCR in Min Pigs

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