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
酶
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]
日期:2003-12-01
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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