CONSTRUCTION OF AN INTERNAL CONTROL TO QUANTITATE MULTIPLE PORCINE CYTOKINE MESSENGER-RNAS BY RT-PCR

A multiple internal control was constructed to be used as an exogenous ly added control in reverse transcription polymerase chain reaction (R T-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 acco mplished by overlapping and extension PCR (OE-PCR) utilizing short oli gonucleotides. The primers were designed to give two products of diffe rent 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 e ndonuclease sites allow construct modification by oligonucleotide addi tion.