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

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 amplif ied together with a known amount of non-homologous competitor cDNA with ide ntical nucleotide primers. The disparate sizes of target and competitor per mitted 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 platted against the initia l amounts of total RNA species used. giving a linear relationship. The slop e of this line was virtually identical with that obtained when the sample R NA was replaced with recombinant target cDNA, indicating that recombinant c DNA 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 varia tion in the final results. the amount of competitor used in the assay was c alculated separately from the equivalence point of the reverse-transcribed total RNA of one of the tissue samples: PCR was performed only for the mini mum number of cycles required to detect products. A standard curve was made in each PCR run by amplifying differing amounts of recombinant cDNA specie s of PT or beta -actin together with a constant amount of its competitor. T he numbers of transcripts in the tissues were then determined directly by P CR incorporating the same amount of respective competitor (as used in the s tandard curve) and comparing the ratios of products with the standard curve . Application of this method revealed that the median ratio of PT message t o 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 deve loped for the quantification of PT transcripts in liver, it can easily be u sed for non-hepatic tissues as well. The technique is simple, quick and sen sitive and requires only a small amount of substrate.