Distinction of mouse interferon-alpha subtypes by polymerase chain reaction utilizing consensus primers and type-specific oligonucleotide probes

• Published in: Journal of interferon research Vol. 14; no. 3; p. 117
• Main Authors: Hughes, Jr, T K, Chin, R, Tyring, S K, Rady, P L
• Format: Journal Article
• Language: English
• Published: United States 01.06.1994
• Subjects: Animals; Base Sequence; Cell Line; Consensus Sequence; DNA Primers; Interferon-alpha - analysis; Mice; Molecular Sequence Data; Oligonucleotide Probes; Polymerase Chain Reaction; RNA - isolation & purification
• ISSN: 0197-8357
• DOI: 10.1089/jir.1994.14.117

We have developed a novel method to study the subtype-specific expression of interferon-alpha (IFN-alpha) in the mouse system: we synthesized and used consensus oligonucleotide primers to allow simultaneous polymerase chain reaction (PCR) amplification of multiple murine IFN-alpha gene sequences. In addition, a set of subtype-specific oligomer probes were designed and used to distinguish between IFN-alpha genes that differ by only a few bases. The consensus primers, corresponding to two regions highly conserved among murine IFN-alpha subtypes, were used in reverse transcription and PCR amplification of total cellular RNA, isolated from IFN-gamma-treated murine L-929 cells, to yield a fragment of the anticipated approximately 520-bp size. Southern analysis of the amplified product using an internal consensus oligomer probe confirmed specific amplification of murine IFN-alpha gene(s). Subtype-specific oligonucleotide probes indicate that IFNs-alpha 1, -alpha 2, and -alpha 5 are present following IFN-gamma treatment, whereas IFN-alpha 4 remains virtually absent. Our results indicate that the expression of specific IFN-alpha subtypes may be subject to complex regulation, dependent upon inducing agents and cell types involved, and countless other factors. The procedure described here represents a novel method for studying the subtleties of the murine IFN-alpha mRNA expression.