Simple, sensitive and accurate method for the quantification of prothrombin mRNA by using competitive PCR.

Abstract

A method for the quantification of prothrombin (PT) mRNA species in hepatic tissues of rats was developed with the use of competitive PCR. To validate the quantification approach, sequential dilutions of total RNA from one of the samples were reverse transcribed. Their equivalent volumes were amplified together with a known amount of non-homologous competitor cDNA with identical nucleotide primers. The disparate sizes of target and competitor permitted the easy identification and quantification of bands in samples after densitometric analysis of ethidium bromide-stained agarose gels. Ratios of intensities of target and competitor bands were plotted against the initial amounts of total RNA species used, giving a linear relationship. The slope of this line was virtually identical with that obtained when the sample RNA was replaced with recombinant target cDNA, indicating that recombinant cDNA behaved in PCR identically with that made by reverse transcription and permitting the estimation of transcripts in reverse transcription reactions by using the recombinant counterpart of each as a standard. To avoid variation in the final results, the amount of competitor used in the assay was calculated separately from the equivalence point of the reverse-transcribed total RNA of one of the tissue samples; PCR was performed only for the minimum number of cycles required to detect products. A standard curve was made in each PCR run by amplifying differing amounts of recombinant cDNA species of PT or beta-actin together with a constant amount of its competitor. The numbers of transcripts in the tissues were then determined directly by PCR incorporating the same amount of respective competitor (as used in the standard curve) and comparing the ratios of products with the standard curve. Application of this method revealed that the median ratio of PT message to beta-actin message in hepatic tissues of 10 normal rats was 0.37, with a mean+/-S.D. of 0.37+/-0.07 (range 0.27-0.47). Although the method was developed for the quantification of PT transcripts in liver, it can easily be used for non-hepatic tissues as well. The technique is simple, quick and sensitive and requires only a very small amount of substrate.