DETERMINATION OF CHANGES IN SPECIFIC GENE-EXPRESSION BY REVERSE TRANSCRIPTION PCR USING INTERSPECIES MESSENGER-RNAS AS INTERNAL STANDARDS

A method is described for the determination of changes in gene express ion by reverse transcription of the target mRNA followed by PCR amplif ication of the resulting cDNA (RT-PCR), using the lipoprotein lipase g ene as the model system. Known proportions of human and rat adipose ti ssue homogenates are mixed and are processed together throughout the R T-PCR procedure so that the rat tissue sen es as an internal standard for the measurement of human adipose tissue lipoprotein lipase (LPL) i n all steps including RNA extraction, reverse transcription and PCR am plification. Taking advantage of the highly conserved sequence of the LPL gene across species, selected homologous regions of the human and rat genes are amplified using the same primer pair and resulting in th e same lengths of amplified DNA fragments. The two amplified products are then separated from each other by making use of differences in the position of a restriction site in the two amplified DNA fragments. Th e method is simple, precise and reproducible and avoids construction o f tailored nucleic acids for use as internal standards.