A SIMPLIFIED, COMPETITIVE RT-PCR METHOD FOR MEASURING RAT IFN-GAMMA MESSENGER-RNA EXPRESSION

We describe an adaptation of competitive RT-PCR to quantitate rat IFN- gamma mRNA expression, An IFN-gamma DNA mimic that shared the same pri mers and had an identical sequence to the target mRNA except for delet ion of 66 nucleotides, was created by a simple PCR amplification from target cDNA. To reduce variations of initial RNA concentrations, beta- actin cDNAs from each target RNA sample were normalized using the dens itometric data. A known amount of pretitrated DNA competitor was then used to analyze the relative levels of target cDNA in different sample s by PCR co-amplification. The amplification efficiency for both targe t and competitor remained constant throughout the PCR reaction, and th e ratio of target to competitor PCR product remained proportional to t he initial ratio of target to competitor. Relative mRNA levels among s amples determined by this method were comparable to levels determined by northern blot analysis. They were also comparable to levels of IFN- gamma protein estimated by ELISA. We conclude that this method can be used to estimate the relative abundance of the target mRNA. This metho d is adaptable to quantitation of other cytokines and is particularly valuable if there are numerous samples, or if the amount of initial mR NA is limited.