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

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• 作者：Richtsteiger ; Ralf ; Henke-Gendo ; Cornelia ; Schmidtke ; Michaela ; Harste ; Gabriele ; Heim ; Albert
• 关键词：Coxsackievirus B3 ; Interferon beta ; Multiplex-PCR ; Poly I ; C transfection ; Quantitative PCR
• 刊名：Cytokine
• 出版年：2003
• 出版时间：December 7, 2003
• 年：2003
• 卷：24
• 期：5
• 页码：190-200
• 全文大小：376 K

文摘

Interferon-β (IFN-β) protein and activity can be detected by enzyme immunoassays and biological assays. However, precise quantification of low IFN-β mRNA concentrations, which is advantageous for investigating IFN-β 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-β 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-β qPCR was utilized to optimize IFN-β induction with dsRNA polyinosic–polycytidylic acid (poly I:C), delivered by a liposomal transfection agent for reproducible but low, non-cell-toxic IFN-β concentrations. For studying coxsackievirus B3 (CVB3) interference with IFN-β expression, CVB3 infected fibroblasts were induced with poly I:C. A significant reduction of IFN-β mRNA but not PBGD mRNA was demonstrated 5 h after CVB3 infection, indicating a specific inhibition of IFN-β expression by CVB3 on the mRNA level, in addition to previously reported effects on the translation/post-translation level. In conclusion, sensitive IFN-β/PBGD multiplex qPCR proved to be a useful tool to study viral interaction with IFN-β expression.