QUANTITATION OF MATRIX GLA PROTEIN MESSENGER-RNA BY COMPETITIVE POLYMERASE CHAIN-REACTION USING GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE ASAN INTERNAL CONTROL

Matrix Gla (gamma-carboxyglutamic acid) protein (MGP) is a vitamin-K-d ependent extracellular matrix protein. A method was developed to quant itate MGP mRNA based on competitive polymerase chain reaction followin g reverse transcription (competitive RT-PCR), The MGP cDNA was coampli fied with a mutant MGP cDNA (competitor). The ratio of MGP to competit or after the PCR reaction was compared to standards to determine the a mount of it MGP mRNA in RT samples. MGP mRNA in as little as 3.125 ng total RNA was accurately quantitated and was far more sensitive than R NA hybridization methods, To control for variations due to sample prep aration, a second competitive RT-PCR was developed to measure the glyc eraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA from the same sample as an internal control, Thus, the amount of MGP is normalized to the amount of the housekeeping gene GAPDH. The accuracy, sensitivity and e ase of this new method enables rapid mRNA quantitation without blottin g, hybridization or autoradiography. The method is particularly advant ageous for MGP mRNA measurement from a small amount of sample. Using t his assay, we established that MGP mRNA increases approx. fivefold wit h co-treatment of retinoic and ascorbic acids.