Figure 5

Expression of TRα and Rev-erbα in potoroo PtK1 cells. A. Endogenous expression of TRα1, TRα2 and Rev-erbα mRNAs was assayed by realtime RT-PCR (left; average of 3 replicas) and conventional PCR (right). Expression of TRα1 and Rev-erbα mRNAs in the opossum, M. domestica (day 0 heads), was measured in a parallel by realtime RT-PCR for comparison. Arrows on photograph at right indicate positions of expected amplification products for each mRNA. Two different primer pairs were targeted against TRα2, which was not observed. The predicted products for TRα1 and Rev-erbα mRNAs were confirmed by sequencing. The Rev-erbα RT-PCR matches sequence obtained from PtK1 cell DNA (additional file 2). No TRα2 mRNA was observed either by realtime RT-PCR or two sets of primers located in exon 7 or 8 and exon 10. B. TRα2 splicing of rat minigenes in PtK1 cells. Rat minigenes with full-length versions of exons 7-10 were expressed inPtK1 cells and assayed by realtime RT-PCR. Two previously characterized single-base point mutations in the 5'ss of exon 9A [32] were analyzed in parallel with wild-type and found to significantly overexpress (+5G) or underexpress (+6G) spliced TRα2 in comparison with wildtype (p < 0.05).