QUANTIFICATION OF MESSENGER-RNA IN HUMAN TISSUE USING FLUORESCENT NESTED REVERSE-TRANSCRIPTASE POLYMERASE CHAIN-REACTION

We report the development of a quantitative nested reverse-transcripta se polymerase chain reaction which utilizes a fluorescence detection s ystem. Using specific primer pairs to study mRNA for endothelin recept ors in the human kidney, we synthesized a cRNA construct containing th e same sequences but yielding a PCR product some 300 base pairs larger than native mRNA. Inclusion of a known amount of construct as interna l standard with tissue RNA prior to cDNA synthesis allowed all reactio ns to occur under the same conditions in the same tube. In the nested PCR reaction, serial dilutions made before the second round enabled co nstruction of a standard curve for each assay, and confirmation that s tandard and sample curves remained parallel. This indicates that both cDNAs amplified at the same rate. One internal primer was fluorescentl y labeled. Quantification of products using an ABI 373A sequencer with Genescan software gave sensitive and reproducible results. Analysis o f a needle biopsy (10 mg) of histologically normal cortex gave 0.4 amo l ET(A) mRNA and 1.6 amol ET(B) mRNA/mu g total RNA. In medulla these values were 0.46 and 1.16 amol/mu g, respectively. Ratios of ET(B) to ET(A) message were 74:26 in cortex and 77:23 in medulla, agreeing with previous ligand binding studies of receptor protein. Intra- and inter assay coefficients of variation were 4.5 and 5.3%. This new method has potential for widespread application to the study of low copy-number mRNA or where only very small amounts of tissue are available, such as biopsy specimens. (C) 1994 Academic Press, Inc.