AN ALTERNATIVE QUANTITATIVE POLYMERASE CHAIN-REACTION METHOD

The exponential nature of the polymerase chain reaction (PCR) makes qu antitation of amplified products possible only if the efficiency of th e enzymatic steps is estimated or cast off. To obtain a relative quant itative measurement of the reverse transcription (RT)-PCR products, a series of seven progressive dilutions achieved by mixing RNA solutions of two different samples to be compared was prepared in a constant fi nal volume. An aliquot of each dilution mix was submitted to a standar d RT-PCR. This range of concentrations allowed the elimination of the tube-to-tube efficiency variations. Indeed, after gel densitometric an alysis of the amplified products, the alignment of the seven measureme nts along a regression line demonstrated that the PCR efficiencies in all tubes was equal allowing a direct comparison between the two sampl es, To illustrate this method, the renal erythropoietin (EPO) expressi on level was compared in anemic and control rats, Reverse transcriptio n was performed using specific primers for EPO and GAPDH genes. The EP O mRNA expression was also checked by Northern blotting, Quantitative BCR indicated that anemic rats produced 19 times more EPO mRNA than di d control rats. The results from Northern blotting matched with those of PCR, This simple new method does not provide absolute amounts of nu cleic acid but relative ones, and it works with any set of primers, It could be used alternatively to methods such as competitive RT-PCR. (C ) 1996 Academic Press, Inc.