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 beta 2 microglobulin (beta 2-m), IL-1, IL-4, IL-6, IL-8, IL-2, IL-10, TNF-alpha, TNF-beta and IFN-gamma. Construction was accomplished by overlapping and extension PCR (OE-PCR) 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.