Quantitative real-time PCR for the measurement of feline cytokine mRNA

We have developed real-time PCR systems to quantitate feline cytokine gene expression. The method is based on the cleavage of fluorescent dye-labelled probes by the 5'-3' exonuclease activity of the Taq DNA polymerase during PCR and measurement of fluorescence intensity by a Sequence Detection Syste m. The feline-specific TaqMan probes were designed to encompass an intron, thus allowing differentiation of complementary DNA versus genomic DNA ampli fication products. Quantitative analysis of cytokine cDNA concentrations wa s performed in comparison to feline GAPDH. Messenger RNA (mRNA) from the un iversally expressed housekeeping gene GAPDH proved to be useful as an ampli fication control and allowed for correction of variations in the efficienci es of RNA extraction and reverse transcription. GAPDH mRNAs were readily de tectable in cDNAs prepared from unstimulated feline peripheral blood mononu clear cells (PBMCs) and from frozen cell pellets, while cytokines (Interleu kin (IL)-4, IL-10, IL-12 p35, IL-12 p40, IFN gamma, IL-16) were expressed a t variable amounts. LFN gamma transcription was found to be upregulated in stimulated PBMCs and feline cell lines. The synthesis of cDNA and the perfo rmance of the PCR in separate tubes proved to be of superior sensitivity co mpared to a single-tube based system. The assays described are highly repro ducible, require no post-PCR manipulation of the amplicons and permit the a nalysis of several hundred PCR reactions per day. With this method it is po ssible to detect and quantify cytokine mRNA expression reliably in small am ounts of cells even after storage of samples for at least 5 years. (C) 1999 Elsevier Science B.V. All rights reserved.