Quantitative multiplex real-time PCR for the sensitive detection of interferon β gene induction and viral suppression of interferon β expression

分子生物学 干扰素 实时聚合酶链反应 多路复用 BETA(编程语言) 信使核糖核酸 生物 基因表达 管家基因 疏孔素原脱氨酶 基因 翻译(生物学) 病毒学 化学 生物化学 生物信息学 程序设计语言 计算机科学 血红素
作者
Ralf Richtsteiger,Cornelia Henke‐Gendo,Michaela Schmidtke,Gabi Harste,Albert Heim
出处
期刊:Cytokine [Elsevier]
卷期号:24 (5): 190-200 被引量:16
标识
DOI:10.1016/j.cyto.2003.09.001
摘要

Interferon-beta (IFN-beta) protein and activity can be detected by enzyme immunoassays and biological assays. However, precise quantification of low IFN-beta mRNA concentrations, which is advantageous for investigating IFN-beta gene expression in small tissue samples or during the early stage of a virus infection, remains a challenge. Therefore, we established a quantitative real-time PCR (qPCR) for IFN-beta and the housekeeping gene porphobilinogen deanimase (PBGD) in separated assays as well as in a multiplex procedure. Sensitivity for both the templates was less than 20 copies with an intra- and interassay variability of less than 5%. IFN-beta qPCR was utilized to optimize IFN-beta induction with dsRNA polyinosic-polycytidylic acid (poly I:C), delivered by a liposomal transfection agent for reproducible but low, non-cell-toxic IFN-beta concentrations. For studying coxsackievirus B3 (CVB3) interference with IFN-beta expression, CVB3 infected fibroblasts were induced with poly I:C. A significant reduction of IFN-beta mRNA but not PBGD mRNA was demonstrated 5 h after CVB3 infection, indicating a specific inhibition of IFN-beta expression by CVB3 on the mRNA level, in addition to previously reported effects on the translation/post-translation level. In conclusion, sensitive IFN-beta/PBGD multiplex qPCR proved to be a useful tool to study viral interaction with IFN-beta expression.
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