PCR-based simultaneous analysis of the interferon-alpha family reveals distinct kinetics for early interferons.

Here we describe a PCR-based analysis system that allows the simple simultaneous assessment of murine interferons (IFN)-alpha and IFN-beta induction in a single reaction. In this analysis, the so-called early IFN-alpha4 can be distinguished from the so-called late IFN-nonalpha4 by employing a primer mixture that amplifies a part of the IFN-alpha genes in which IFN-alpha4 exhibits a deletion of 15 nucleotides compared to IFN-nonalpha4. By including a final denaturation and a slow cooling step at the end of the PCR procedure, hybrid formation was avoided that regularly occurred when standard protocols were used. Separation of the amplification products on 4.5% agarose gels allowed the comparative assessment of the classical type I IFNs. Using this analysis system, we could show that in immortalized adult fibroblasts, IFN-beta is induced first and the two types of IFN-alpha are induced later and simultaneously. When similar fibroblasts derived from mice that lack IFN-beta were tested, the IFN response was delayed. However, now IFN-alpha4 appeared first and apparently induced the cascade of IFN-nonalpha4. This confirms the role of IFN-beta as master regulator of the normal IFN response.