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

The Biochemical Journal
P K GroverR L Ryall

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...Continue Reading

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Citations

Mar 26, 2003·Brain Research. Molecular Brain Research·Estel Van der GuchtLutgarde Arckens
Aug 8, 2009·Bioscience, Biotechnology, and Biochemistry·Fumie ShinomiyaTakeshi Kawahara
Jun 3, 2008·Reproduction : the Official Journal of the Society for the Study of Fertility·Laura F PisaniFulvio Gandolfi
Oct 30, 2007·Reproduction : the Official Journal of the Society for the Study of Fertility·Fabiana CilloFulvio Gandolfi

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