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

AbstractInterferon-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.
AuthorsRalf Richtsteiger, Cornelia Henke-Gendo, Michaela Schmidtke, Gabriele Harste, Albert Heim (Affiliation: Institute for Virology, Hannover Medical School, Hannover, Germany.)
JournalCytokine (Cytokine) Vol. 24 Issue 5 Pg. 190-200 (Dec 7 2003) ISSN: 1043-4666 [Print] United States
PMID14596815 (Publication Type: Journal Article)
Chemical References
  • RNA, Messenger
  • Receptors, Virus
  • coxsackievirus B receptor
  • Interferon-beta
  • Hydroxymethylbilane Synthase
Topics
  • Gene Expression Regulation (physiology)
  • Humans
  • Hydroxymethylbilane Synthase (metabolism)
  • Interferon-beta (biosynthesis, genetics)
  • RNA, Messenger (metabolism)
  • Receptors, Virus (metabolism)
  • Reverse Transcriptase Polymerase Chain Reaction
  • Temperature
  • Time Factors
  • Transcriptional Activation
  • Virus Diseases (metabolism)

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