Differential expression of IFN-alpha subtypes in human PBMC: evaluation of novel real-time PCR assays.
Studies of the human IFN-alpha subtype system have been hampered by the lack of efficient procedures to quantify and differentiate the expression of the highly homologous IFN-alpha subtypes. Here we evaluate four novel real-time PCR assays for the specific detection and quantification of IFN-alpha mRNA for the subtypes alpha(2), alpha(6), alpha(8) and alpha(1/13) in a combined assay in human peripheral blood mononuclear cells (PBMC). This included (a) the selection of beta-glucuronidase (GUS) as a suitable housekeeping gene for relative quantification; (b) verification of the specificity by using human DNA of different IFN-alpha subtypes; and (c) comparison of the amplification efficiencies among the different assays. This highly sensitive method allows the detection of low-level, constitutive IFN-alpha mRNA and shows differences in the composition of constitutive IFN-alpha subtypes compared to other cell types (HeLa and HEp-2). The in vitro stimulation of PBMC with Newcastle disease virus (NDV), Respiratory syncytial virus (RSV) or an inactivated Herpes simplex (HSV) preparation leads to the transcriptional induction of all IFN-alpha subtypes investigated but to different expression levels. Among the subtypes detected, IFN-alpha(13/1) and alpha(2) are the major transcripts followed by alpha(8), and finally alpha(6) as a minor transcribed subtype. Time-kinetics of IFN-alpha transcriptional activation also revealed variations in the course of IFN-alpha transcription between NDV, RSV or HSV. The data obtained from the real-time PCR assays correlated well with IFN-alpha(2) protein release. In conclusion, we have demonstrated the suitability and reliability of new real-time PCR assays for the rapid and efficient analysis of IFN-alpha subtype expression.
Ruhr-University Bochum, Experimental Pneumology, University Hospital Bergmannsheil, BGFA XU 19, Bürkle-de-la-Camp-Platz 1, D-44789, Bochum, Germany. stefan.loeseke@ruhr-uni.bochum.de
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Löseke
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Grage-Griebenow
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Wagner
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Gehlhar
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Bufe
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D017931:DNA Primers; D015342:DNA Probes; D005966:Glucuronidase / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D016898:Interferon-alpha / Q000096:biosynthesis* / Q000145:classification* / Q000235:genetics; D007372:Interferons / Q000096:biosynthesis / Q000235:genetics; D007700:Kinetics; D007963:Leukocytes, Mononuclear / Q000276:immunology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D017931:DNA Primers; D015342:DNA Probes; C554164:IFNA8 protein, human; D016898:Interferon-alpha; D007372:Interferons; D005966:Glucuronidase