Figure 6.

Expression of CYP24 and P4501α mRNAs in the kidney of each rat model. Radioautography of RT-PCR products of CYP24 and P4501α mRNAs in microdissected PCT. Three-week-old rats were fed a vitamin D-replete or a vitamin D-deficient low Ca diet for 2 weeks. Microdissection and RT-PCR were performed as described (18, 19) with a slight modification. Final volume of the PCR reaction was 20 μl, and composition of the solution was 10 mM Tris⋅HCl (pH 8.3), 50 mM KCl, 2 mM MgCl₂, gelatin 0.01%, 200 nM sense and antisense primers, 0.1 mM dNTPs, 74 kBq of [³³P]dCTP, and 1 unit of Taq DNA polymerase. The program of PCR was as follows: 25 cycles of 94°C for 40 sec, 50°C for 1 min, and 72°C for 1 min. The expected size of RT-PCR products for P4501α and CYP24 were 253 and 482 bp, respectively.