Quantitation of vWF mRNA by competitive RT-PCR. The approach used to quantitate vWF mRNA in murine tissues is shown in this diagram. (A) The synthetic plasmid used in these experiments is shown. It contains upstream and downstream sequences from several hemostatic genes including vWF. The plasmid was linearized with Kpn I and used as a template for in vitro transcription with T7 RNA polymerase. The resulting cRNA standard was then used as a competitor during RT-PCR. (B) One microgram of total tissue RNA and a fixed amount of the cRNA standard were mixed and then reverse transcribed into cDNA. The resulting mixture thus contained cDNA transcribed from both tissue mRNA and the standard cRNA. Serial twofold dilutions of the DNA mixture were amplified (30 cycles) using the vWF-specific primers containing 1 × 106 cpm of 32P-end labeled sense primer. (C) The PCR reaction mixture was electrophoresed through 2% agarose gels and photographed. The size difference between the PCR products permits easy separation of the standard (172 bp) from the target (1,130 bp). The appropriate bands for each PCR product were cut out of the gel and the radioactivity in each was determined using a scintillation counter. (D) The cpm in the PCR products obtained from the cRNA standard was plotted against the number of cRNA standard molecules used for RT-PCR using a double logarithmic scale. The cpm in the PCR product from the target mRNA was plotted against the amount of target RNA used for RT-PCR. Because the reaction rates for the cRNA standard and target mRNA should be the same, the number of vWF mRNA molecules in the tissue can be determined by extrapolation using the standard cRNA curve.
