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 qRT-PCR
赵璐 1冯烨 1张森 1陈月红 1李靖 1李裕昌 1姜涛1
作者信息
- 1. 军事科学院军事医学研究院,病原微生物生物安全国家重点实验室,北京 100071
- 折叠
摘要
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.
Abstract
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 A Ct 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.
关键词
内参基因/流感病毒/干扰素/逆转录定量PCR/A549细胞/THP-1细胞Key words
reference gene/influenza virus/interferon/reverse transcription-quantitative PCR/A549 cell/THP-1 cell引用本文复制引用
基金项目
病原微生物生物安全国家重点实验室自主研究课题(SKLPBS2209)
病原微生物生物安全国家重点实验室自主研究课题(SKLPBS2120)
出版年
2024
国家科技期刊平台
NSTL
万方数据