PCR-Based Simultaneous Analysis of the Interferon-Alpha Family Reveals Distinct Kinetics for Early Interferons

Abstract

Here we describe a PCR-based analysis system that allows the simple simultaneous assessment of murine interferons (IFN)-α and IFN-β induction in a single reaction. In this analysis, the so-called early IFN-α4 can be distinguished from the so-called late IFN-nonα4 by employing a primer mixture that amplifies a part of the IFN-α genes in which IFN-α4 exhibits a deletion of 15 nucleotides compared to IFN-nonα4. 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-β is induced first and the two types of IFN-α are induced later and simultaneously. When similar fibroblasts derived from mice that lack IFN-β were tested, the IFN response was delayed. However, now IFN-α4 appeared first and apparently induced the cascade of IFN-nonα4. This confirms the role of IFN-β as master regulator of the normal IFN response.