Quantification of mRNA in human tissue using fluorescent nested reverse-transcriptase polymerase chain reaction.

Abstract

We report the development of a quantitative nested reverse-transcriptase polymerase chain reaction which utilizes a fluorescence detection system. Using specific primer pairs to study mRNA for endothelin receptors in the human kidney, we synthesized a cRNA construct containing the same sequences but yielding a PCR product some 300 base pairs larger than native mRNA. Inclusion of a known amount of construct as internal standard with tissue RNA prior to cDNA synthesis allowed all reactions to occur under the same conditions in the same tube. In the nested PCR reaction, serial dilutions made before the second round enabled construction of a standard curve for each assay, and confirmation that standard and sample curves remained parallel. This indicates that both cDNAs amplified at the same rate. One internal primer was fluorescently labeled. Quantification of products using an ABI 373A sequencer with Genescan software gave sensitive and reproducible results. Analysis of a needle biopsy (10 mg) of histologically normal cortex gave 0.4 amol ETA mRNA and 1.6 amol ETB mRNA/micrograms total RNA. In medulla these values were 0.46 and 1.16 amol/micrograms, respectively. Ratios of ETB to ETA message were 74:26 in cortex and 77:23 in medulla, agreeing with previous ligand binding studies of receptor protein. Intra- and interassay 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.