Quantitative multiplex real-time PCR for the sensitive detection of interferon beta gene induction and viral suppression of interferon beta expression.
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.
Institute for Virology, Hannover Medical School, Hannover, Germany.
Ralf
R
Richtsteiger
Cornelia
C
Henke-Gendo
Michaela
M
Schmidtke
Gabriele
G
Harste
Albert
A
Heim
D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D011163:Hydroxymethylbilane Synthase / Q000378:metabolism*; D016899:Interferon-beta / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D011991:Receptors, Virus / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013696:Temperature; D013997:Time Factors; D015533:Transcriptional Activation; D014777:Virus Diseases / Q000378:metabolism
D012333:RNA, Messenger; D011991:Receptors, Virus; C105941:coxsackievirus B receptor; D016899:Interferon-beta; D011163:Hydroxymethylbilane Synthase