[Quantification of mRNA using the polymerase chain reaction--cytokines in rheumatoid arthritis]

Abstract

A method for quantitation of specific mRNA species by the polymerase chain reaction (PCR) has been developed by using a synthetic RNA as an internal standard. The specific target mRNA and the internal standard are co-amplified in one reaction in which the same primers are used. The amount of mRNA is then quantitated by extrapolating against the standard curve generated with the internal standard. The synthetic internal standard RNA consists of a linear array of the sequence of upstream primers of multiple target genes followed by the complementary sequences to their downstream primers in the same order. This quantitative PCR method provides a rapid method of quantifying the amount of a specific mRNA in a sample of less than 0.1 ng of total RNA. In addition, the same internal standard RNA is used, with appropriate primer pairs, to quantitate multiple different mRNA species in parallel. This technique affords a very sensitive means of measuring important regulatory cytokines in the local inflammatory tissue of rheumatoid arthritis (RA).