中华鳖实时定量PCR内参基因的筛选与评价

Screening and Evaluation of qPCR Reference Genes in Pelodiscus sinensis

选择合适的内参基因是采用q-PCR方法研究基因表达的前提.选取中华鳖GAPDH、EF1α、18SrRNA、Tubulin和β-actin等5个候选内参基因,通过q-PCR方法得到各基因在不同组织中的Ct值,利用4种内参筛选软件和综合评定法进行评价.geNorm和NormFinder软件分析均显示18SrRNA>EF1α>β-actin>GAPDH>Tubulin(稳定性由高到低).BestKeeper软件分析显示18SrRNA>GAPDH>EF1α>β-actin>Tubulin.RefFinder软件分析和综合评价法显示18SrRNA最佳.推荐优先使用18SrRNA或EF1α,不建议用β-actin和Tubulin.上述结果可为中华鳖和其它爬行动物的基因表达研究提供参考依据.

Selection of reference genes was important to the study of gene expression via qPCR method. GAPDH ,EF1a, 18S rRNA,Tubulin and i3-actin of Chinese softshell turtle Pelodiscus sinensis were selected as the candidate reference genes. Cycle threshold value （Ct） of different tissues were obtained through qPCR method,and the Ct value were further analyzed via 4 softwares and the comprehensive evaluation method on reference gene evaluation. The stability result of both geNorm and NormFinder was 18S rRNA~ EFla〉 fl-actinD GAPDH~ Tubulin （From high to low）. The result of BestKeeper was 18S rRNA〉 GAPDH〉 EF1 a〉13-actin〉 Tubulin. The result of RefFinder and comprehensive evaluation method showed that 18S rRNA was the best reference gene. There- fore, 18S rRNA or EF1α was recommend as the reference gene of P. sinensis,instead of β-actin and Tubulin. The study could provide a guidance and reference for the studying of gene expression of Chinese softshell turtle and other related reptiles.