实时荧光定量PCR筛选鸡基因表达最佳内参基因

Screening optimum reference gene for gene expression in chicken by quantitative real-time PCR

为筛选在鸡基因表达时稳定表达的内参基因,本试验以6、14、22及30周龄的地方品种固始鸡与6周龄的商业品种罗斯308肉鸡为试验动物,以B2M、28S、GAPDH、β-actin及HSP70为候选内参基因,利用qRT-PCR对这5个候选内参基因的相对表达量进行测定,通过独立评价软件geNorm、NormFinder、SPSS和Ct值分析法筛选出在不同组织、不同发育时期和不同品种间稳定表达的最佳内参基因。结果表明:在鸡不同组织中,28S为最佳内参基因;在鸡不同发育时期中,GAPDH为最佳内参基因;在不同品种鸡中最佳内参基因组合为GAPDH和28S。综上所述,在以地方品种固始鸡为例的不同组织,不同时期间最佳内参基因分别为28S和GAPDH;以地方品种固始鸡和商业品种罗斯308肉鸡为例的不同品种间最佳内参基因为GAPDH+28S基因组合。综上,本研究筛选出了鸡不同试验条件下的最佳内参基因,为规范鸡基因定量表达分析中内参基因的使用提供参考依据。

In order to screen the internal reference genes stably expressed in chicken,the local Gushi chicken of 6,14,22,and 30-week-old and the commercial variety Ross 308 of 6-week-old were used as experimental animals.Using B2 M,28 S,GAPDH,β-actin and HSP70 as candidate reference genes,the relative expression levels of these five candidate reference genes were determined by qRT-PCR.by the three softwares(geNorm,NormFinder and SPSS)and Ct value analysis.The best internal reference genes were screened out in different tissues,different development stages and different varieties.The results showed that 28 s was the best internal reference gene in different tissues of chicken;GAPDH was the best internal reference gene in different development stages;GAPDHand 28 s were the best combination of internal reference genes in different chicken varieties.In summary,the best internal reference genes under experimental conditions were identified in this study,which provided a reference for standardizing the use of internal reference genes in chicken genes for quantitative expression analysis.