Quantitation of porcine cytokine and beta 2-microglobulin mRNA expression by reverse transcription polymerase chain reaction.

摘要

A quantitative reverse transcription polymerase chain reaction (Q-RT-PCR) method was developed to measure pig cytokine mRNA expression. The method utilized an internal control with primer sequences for interleukin (IL)-1alpha, 2, 4, 6, 8, 10, tumor necrosis factor (TNF)-alpha, TNF-beta, interferon (IFN)-gamma and beta-2 microglobulin (beta(2)-m). The control was modified by insertion of sequences for IL-12 (p35 and p40). Pig blood mononuclear cells (BMCs) were stimulated in vitro with phytohemagglutinin-P (PHA-P) or bacterial lipopolysaccharide and cytokine or beta(2)-m mRNA quantified. To evaluate method performance and the use of beta(2)-m as a housekeeping gene (HKG), beta(2)-m mRNA expression was examined. Quantitative analysis was achieved at up to threefold differences between control and target for beta(2)-m. Results were reproducible with coefficients of variations (CVs) ranging between 12.5% and 22.4%. There were no significant differences in beta(2)-m mRNA between treated and untreated cells or between untreated cells of three pigs (p>/=0.05) suggesting that beta(2)-m can be used as a HKG. The method allows quantitation of multiple cytokine mRNAs using a single internal control subjecting target and control to the same conditions throughout the Q-RT-PCR. The system is versatile since the control plasmid can be modified by insertion or deletion of sequences.