COMPETITIVE RNA TEMPLATES FOR DETECTION AND QUANTITATION OF GROWTH-FACTORS, CYTOKINES, EXTRACELLULAR-MATRIX COMPONENTS AND MATRIX METALLOPROTEINASES BY RT-PCR

Detection of low-abundance mRNAs by reverse transcription-polymerase c hain reaction (RT-PCR) has become a standard technique to determine ge ne expression by tissues and cells in culture. The ability to determin e relative or absolute copy number of specific mRNAs has been difficul t due to inadequate internal standards to control for sample-re-sample variation. The use of a synthetic RNA standard with identical sequenc es to the PCR primers allows reproducible quantitation between samples and assays. By designing multi-sequence templates several specific mR NAs can be quantitated using a single template. Addition of multiple t emplates to a single RT reaction allows the quantitation of a large nu mber of targets from as little as 4 mu g of total RNA. In this report, we present a series of seven primer/template systems to detect and qu antitate 52 specific messages, including 26 growth factors and recepto rs, 8 extracellular matrix components, 10 matrix-modifying enzymes and their inhibitors and 8 cytokines.