双酚A诱导下双齿围沙蚕实时荧光定量PCR内参基因的筛选

Reference Gene Selection for Quantitative Real-Time PCR in Perinereis aibuhitensis under BPA Exposure

为了筛选出在双酚A诱导下双齿围沙蚕(Perinereis aibuhitensis)体壁组织中最适内参基因,以不同浓度双酚A诱导下双齿围沙蚕互补脱氧核糖核酸(cDNA)为模板进行实时荧光定量PCR,利用GeNorm、NormFinder和Bestkeeper三个软件对肌动蛋白(beta-actin,Actin)、核糖体蛋白5(ribosomal protein L5,RPL5)、甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)、微管蛋白(β-tubulin,Tub)、琥珀酸脱氢酶(succinate dehydrogenase complex flavoprotein subunit A,SDHA)和组蛋白(Histone Cluster 1 H2A Family Member A,HH2A)这6个备选内参基因的表达量进行分析,筛选出稳定表达的内参基因。结果表明6个内参基因的基因表达量由大到小为Actin>GAPDH>SDHA>HH2A>Tub>RPL5,Bestkeeper软件分析得到6种内参基因稳定性排名为Actin>RPL5>SDHA>Tub>HH2A>GAPDH,GeNorm软件分析得出6种内参基因的稳定性排名为SDHA>RPL5>Actin>Tub>HH2A>GAPDH,Normfinder分析的结果为Actin>SDHA>RPL5>Tub>HH2A>GAPDH。综合以上结果确定双酚A诱导下双齿围沙蚕最优内参基因为Actin,不推荐GAPDH作为内参基因使用。推荐同时选取多对内参基因进行实验,可以进一步减少误差。

In order to select appropriate reference genes for fluorescent quantitative real time PCR in Perinereis aibuhitensis exposed to bisphenol A (BPA) with different concentrations,six candidate reference genes including beta-actin (Actin),ribosomal protein L5 (RPL5),glyceraldehyde-3-phosphate dehydrogenase (GAPDH),β-tubulin (Tub),succinate dehydrogenase complex flavoprotein subunit A (SDHA) and Histone Cluster 1 H2A Family Member A (HH2A) were analyzed by Bestkeeper,GeNorm and Normfinder softwares.The gene expression levels of these candidate genes were Actin > GAPDH > SDHA > HH2A > Tub > RPL5.The Bestkeeper software analysis indicated that the stability of these genes was Actin > RPL5 > SDHA > Tub > HH2A > GAPDH.The GeNorm software analysis indicated that the stability of these genes was SDHA > RPL5 > Actin > Tub > HH2A > GAPDH.The Normfinder software analysis indicated that the stability of these genes was Actin > SDHA > RPL5 > Tub > HH2A > GAPDH.To sum up,the best reference gene for fluorescent quantitative real time PCR in P.aibuhitensis under BPA exposure was Actin,in the meanwhile,GAPDH was not recommended as the reference gene.Furthermore,using multiple reference genes can improve the accuracy of experiment.