Quantitation of porcine cytokine and beta 2-microglobulin mRNA expression by reverse transcription polymerase chain reaction.
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.
Reddy
N R
NR
Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada.
Wilkie
B N
BN
D000818:Animals; D001483:Base Sequence; D016207:Cytokines / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D005069:Evaluation Studies as Topic; D015870:Gene Expression; D018664:Interleukin-12 / Q000235:genetics; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000276:immunology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008969:Molecular Sequence Data; D010835:Phytohemagglutinins / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics*; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity; D013552:Swine; D001613:beta 2-Microglobulin / Q000235:genetics*
D016207:Cytokines; D017931:DNA Primers; D008070:Lipopolysaccharides; D010835:Phytohemagglutinins; D012333:RNA, Messenger; D001613:beta 2-Microglobulin; C028784:phytohemagglutinin-P; D018664:Interleukin-12