A reverse transcription-polymerase chain reaction method to analyze porcine cytokine gene expression.
A reverse transcription-polymerase chain reaction (RT-PCR) method was developed in order to provide a highly sensitive, rapid, and simple means of simultaneously measuring the expression of porcine cytokines in immune cell populations. Oligonucleotide primers were designed to amplify porcine cytokine cDNA from genes encoding IL-1 alpha, IL-1 beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IFN-gamma, TNF-alpha, TNF-beta and the housekeeping genes beta-actin and cyclophilin by PCR. Primers were chosen from different exons to detect for possible genomic DNA contamination of samples. To validate RT-PCR, unstimulated and concanavalin A (ConA) stimulated porcine peripheral blood mononuclear cells (PBMCs) were cultured from 2 h to 72 h, RNA was extracted and reverse transcribed, and cDNA was amplified using the different primer sets. Band intensities of PCR products were quantified by densitometric scanning and values were normalized against cyclophilin. For each of the cytokines, the kinetics of gene expression were similar among PBMCs isolated from different animals and could be grouped into two main patterns. Lymphocyte derived cytokines (IL-2, IL-4, IFN-gamma, and TNF-beta) exhibited low level expression in unstimulated cells and increased expression in ConA-stimulated PBMCs. IFN-gamma and IL-2 mRNA levels peaked at 24 h and returned to baseline by 72 h, whereas IL-4 and TNF-beta mRNA levels did not return to baseline by 72 h. In contrast, substantial mRNA levels for inflammatory cytokines (IL-1 alpha, IL-1 beta, IL-6, IL-8, IL-12, and TNF-alpha) and IL-10 were detected from both unstimulated and ConA-stimulated PBMCs. Results indicate that RT-PCR is a sensitive and convenient method to monitor cytokine mRNA expression in porcine samples.
Dozois
C M
CM
Laboratoire de Pharmacologie Toxicologie, INRA, Toulouse, France.
Oswald
E
E
Gautier
N
N
Serthelon
J P
JP
Fairbrother
J M
JM
Oswald
I P
IP
D000199:Actins / Q000235:genetics; D000818:Animals; D016207:Cytokines / Q000235:genetics*; D008297:Male; D019696:Peptidylprolyl Isomerase / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*; D013552:Swine / Q000276:immunology*
D000199:Actins; D016207:Cytokines; D012333:RNA, Messenger; D019696:Peptidylprolyl Isomerase