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 versatile method for the analysis of human and murine cytokine mRNA expression. This paper describes for the first time a reverse transcription-polymerase chain 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-18. The main features of the methodology are: (1) a unique RT for all quantifications, (2) the addition of homologous DNA internal controls (IC) of equal length to the corresponding cytokine and consequently co-amplification of the target cytokine and the IC with equivalent efficacy, (3) PCR and detection of amplicons for all cytokines simultaneously, (4) cytokine quantification in relation to a housekeeping gene control (glyceraldehyde-3-phosphate dehydrogenase, GAPDH), (5) detection of the amplicons by enzyme linked immunosorbent assay (ELISA) using a chemiluminescent substrate with high sensitivity and wide dynamic range, (6) automation of the detection system for analysis of a large number of samples. This highly sensitive quantitative RT-PCR assay (able to detect 100-200 cytokines mRNA copies/75x10(3) cells) was validated on peripheral blood mononuclear cells (PBMC) from pigs infected or not with pseudorabies virus (PRV), re-stimulated in vitro by a mitogen or antigens.
Dufour
V
V
Service de Biologie Moléculaire, AFSSA, BP53, 22 440 Ploufragan, France. v.dufour@ploufragan.afssa.fr
Arnauld
C
C
Lantz
O
O
Peguillet
I
I
Morvilliers
K
K
Emmanuel
A
A
Jestin
A
A
D000818:Animals; D001483:Base Sequence; D016207:Cytokines / Q000032:analysis / Q000235:genetics*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000032:analysis / Q000235:genetics; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013552:Swine
D016207:Cytokines; D012333:RNA, Messenger; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases