A COMPETITIVE RT-PCR METHOD FOR THE QUANTITATIVE-ANALYSIS OF CYTOKINEMESSENGER-RNAS IN MOUSE-TISSUES

The authors describe the design and validation of a competitive RT-PCR method for the efficient and reproducible quantitation of mRNA molecu les of IFN-gamma, TNF-alpha, IL-4 and IL-IO in mouse spleen RNA extrac ts, Before being subjected to RT-PCR, the RNA extracts were supplement ed with internal control RNAs (IC-RNAs), which were constructed by ins erting DKA fragments in the cDNA of the respective cytokines. The effi ciency of amplification of the target and the IC-RNA was shown to rema in equal over a wide range of cycle numbers, Reproducibility was such that differences in mRNA contents that were greater than 17% could be detected between two RNA samples run in parallel, Normal mouse spleen tissue was found to contain 10(7)-10(8) molecules of TNF-alpha, IFN-ga mma, IL-4 and IL-IO mRNA per pg total RNA extracted, Injection of anim als with anti-CD3 antibody, a well-known cytokine inducer, resulted in a moderate increase in TNF-alpha and IL-10 mRNA levels (14- and 24-fo ld, respectively), and in a substantially greater increase in the leve ls of mRNA for IL-4 and IFN-gamma (199- and 851-fold, respectively), T hese results demonstrate an accurate and reliable quantitation of cyto kine mRNA levels in animal tissues. (C) 1997 Academic Press Limited.