Quantification of matrix protein mRNAs expression during mineralized tissue formation in rat marrow cell culture by a real time quantitative PCR method

We applied a real time quantitative PCR method with a fluorogenic probe to quantify mRNA expression of bone matrix proteins during osteoblastic differentiation of cultured bone marrow mesenchymal cells. After trypsinization, cells were subcultured at 10 x 10(4)/35 mm cb well in the presence of 15% fetal bovine serum, dexamethasone (Dex), beta -glycerophosphate (beta -GP), and ascorbic acid phosphate. At day 3, 9 and 12 in subculture, total RNA (2 mug) extracted from each well was reverse-transcribed into cDNA with AMV reverse transcriptase XL. To measure rat alkaline phosphatase (ALP), osteopontin (OP) and bone Gla-protein (BGP, or osteocalcin) mRNA levels, the quantitative PCR was performed using respective primer pair and fluorogenic probe. House keeping gene of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was also measured as an internal standard. Visible mineralized nodule formation began to appear at 9 days, when apparent BGP mRNA was detected. High ALP mRNA level was recognized even at 6 days. The levels of these bone specific mRNAs increased as time passed. In contrast to these findings, subculture done under the same conditions except for the lack of Dex did not show mineralized nodules, nor did they show high levels of ALP and BGP mRNA expressions. Quantification of osteoblastic phenotypic mRNA by the quantitative PCR method is especially attractive in regard to high specificity and sensitivity, which is 10(5) times higher than that of Northern blot analysis. As the differentiation can occur on the surface of bioactive ceramics, the quantitative PCR method is a powerful and straightforward technique to elucidate the cells/ceramics interaction.