Construction of an Internal Control to Quantitate Multiple Porcine Cytokine mRNAs by RT-PCR

Abstract

A multiple internal control was constructed to be used as an exogenously added control in reverse transcription polymerase chain reaction (RT-PCR) for pig cytokines. It consists of 5′ and 3′ primer sequences in the order of β2 microglobulin (β2-m), IL-1, IL-4, IL-6, IL-8, IL-2, IL-10, TNF-α, TNF-β and IFN-γ. Construction was accomplished by overlapping and extension PCR (OEPCR) utilizing short oligonucleotides. The primers were designed to give two products of different sizes on co-amplification of control and target RNAs by RT-PCR in a single tube. This permits analysis of message for several cytokines using a single exogenously added competitive template. Incorporated endonuclease sites allow construct modification by oligonucleotide addition.