新西兰白兔不同内脏组织中实时荧光定量PCR内参基因的筛选

Screening reference genes by qRT-PCR in different visceral tissues of New Zealand white rabbits

【目的】筛选新西兰白兔不同内脏组织中稳定表达的实时荧光定量PCR(quantitative real-time PCR,qRTPCR)内参基因,为新西兰白兔基因定量表达分析中内参基因的规范使用提供参考。【方法】以70日龄新西兰白兔的心脏、肝脏、脾脏、肺脏、肾脏及胃6种组织为研究对象,以β-actin、GAPDH、Ywhaz、HPRT1、18S rRNA、Sdha、B2M和CYP为候选内参基因,利用qRT-PCR和在线内参基因稳定性评价工具RefFinder中的geNorm、NormFinder和BestKeeper程序对候选内参基因的表达稳定性进行分析。【结果】新西兰白兔不同内脏组织中内参基因稳定性不同,心脏组织中最稳定表达的内参基因为β-actin和HPRTI,肝脏组织中最稳定表达的内参基因为HPRT1和GAPDH,脾脏组织中最稳定表达的内参基因为β-actin和GAPDH,肺脏组织中最稳定表达的内参基因为Sdha和18S rRNA,肾脏和胃组织中最稳定表达的内参基因为CYP和β-actin。【结论】根据筛选出的新西兰白兔不同内脏组织中对应的稳定内参基因情况,推荐选择表达最稳定的3个及以上内参基因来进行目的基因的归一化,以提高真实性和可靠性。

【Objective】The aim of this study was to screen stably expressed qRT-PCR reference genes in different organs of New Zealand white rabbits,and to provide reference for standardizing the use of internal reference genes in quantitative gene expression analysis of New Zealand white rabbits.【Method】In this study,six tissues of heart,liver,spleen,lung,kidney and stomach of 70-day-old New Zealand White rabbits were used as research object.β-actin,GAPDH,Ywhaz,HPRT1,18S rRNA,Sdha,B2M and CYP were used as candidate reference genes,and the expression stability of the candidate reference genes were analyzed using qRT-PCR technique and geNorm,NormFinder and BestKeeper programs in the online tool RefFinder.【Result】The stability of internal reference genes of New Zealand white rabbits was different in different visceral tissues.The most stable reference genes wereβ-actin and HPRT1 in heart tissues,HPRT1 and GAPDH in liver tissues,β-actin and GAPDH in spleen tissues,Sdha and 18S rRNA in lung tissues,and CYP andβ-actin in kidney and stomach tissues.【Conclusion】According to the corresponding stable internal reference genes screened out from different visceral tissues of New Zealand white rabbits,it is recommended to select the three or more most consistently expressed reference genes for the normalization of target genes to improve authenticity and reliability.