适用于流感病毒感染研究的qRT-PCR内参基因评估

Evaluation of reference genes under influenza virus infection for qRTPCR

目的确定稳定的内参基因以比较病毒感染下靶标基因的转录水平,为研究宿主⁃流感病毒相互作用提供参考。方法在经典流感感染细胞模型(A549和THP⁃1细胞)中使用逆转录定量实时PCR(RT⁃qPCR)检测6个候选内参基因[甘油醛⁃3⁃磷酸脱氢酶(GAPDH)、β⁃肌动蛋白(β⁃actin)、18S RNA、β2微球蛋白(B2M)、泛素结合酶E2D2(UBE2D2)、核糖体蛋白L37A(RPL37A)]的相对表达量。使用BestKeeper、GeNorm、NormFinder和比较ΔCt法等方法综合评估内参基因的稳定性。结果在不同条件下,内参基因稳定性存在差异。当综合考虑流感病毒感染和免疫激活等因素时,β⁃actin和GAPDH分别是A549细胞和THP⁃1细胞中最稳定的内参基因,其次是UBE2D2和B2M。结论鉴定出的A549和THP⁃1细胞在流感病毒感染、干扰素及脂多糖处理时表达内参基因的稳定性及优选内参基因,对病毒感染机制研究具有借鉴意义。

Objective To identify stable reference genes for a comparison of the transcription levels of target host genes under viral infection in order to provide data for studies on interactions between the host and the influenza virus.Methods Reverse transcription quantitative real⁃time PCR(RT⁃qPCR)was performed to detect the relative expression levels of six candidate reference genes,including glyceraldehyde 3⁃phosphate dehydrogenase(GAPDH),β⁃actin,18S RNA,β2⁃microglobulin(B2M),ubiquitin⁃conjugating enzyme E2D2(UBE2D2),and ribosomal protein L37A(RPL37A)in classical cell models(A549 cells and THP⁃1 cells)under different conditions.The stability of the reference genes was evaluated using such methods as BestKeeper,GeNorm,NormFinder,and comparative ΔCt method.Results The stability of reference genes varied depending on conditions.When such experimental factors as influenza virus infection and immune activation were taken into consideration,β⁃actin and GAPDH were identified as the most stable reference genes in A549 cells and THP⁃1 cells,followed by UBE2D2 and B2M.Conclusion The optimal reference genes in A549 cells and THP⁃1 cells under influenza virus infection or after being treated with interferons or LPS have been identified,which is of referential value for studying the mechanisms of viral infections.