Quantification of porcine cytokine gene expression using RT-PCR, a homologous internal control and chemiluminescence for microplate detection

The polymerase chain reaction (PCR) has proved to be a sensitive and versat ile method for the analysis of human and murine cytokine mRNA expression. T his paper describes for the first time a reverse transcription-polymerase c hain reaction (RT-PCR) at end-point for the quantification of five porcine cytokines: interferon (IFN)-gamma, interleukin (IL)-2, IL-4, IL-10 and IL-1 8. The main features of the methodology are: (1) a unique RT for all quanti fications, (2) the addition of homologous DNA internal controls (IC) of equ al length to the corresponding cytokine and consequently co-amplification o f the target cytokine and the IC with equivalent efficacy, (3) PCR and dete ction of amplicons for all cytokines simultaneously, (4) cytokine quantific ation in relation to a housekeeping gene control (glyceraldehyde-3-phosphat e dehydrogenase, GAPDH), (5) detection of the amplicons by enzyme Linked im munosorbent assay (ELISA) using a chemiluminescent substrate with high sens itivity and wide dynamic range, (6) automation of the detection system for analysis of a large number of samples. This highly sensitive quantitative R T-PCR assay (able to detect 100-200 cytokines mRNA copies/75 x 10(3) cells) was validated on peripheral blood mononuclear cells (PBMC) from pigs infec ted or not with pseudorabies virus (PRV), re-stimulated in vitro by a mitog en or antigens. (C) 1999 Elsevier Science B.V. All rights reserved.