Figure 3
Expression of TRα1, Rev-erbα and TRα2 mRNAs in the opossum Monodelphis domestica. A. Expression of TRα1 and Rev-erbα in staged animals is shown for heads and bodies (0 days and one week of age, hatched bars) or cerebral cortex and liver (2-18 weeks, evenly shaded bars) based on realtime RT-PCR analysis. Results are averages from at least three animals with each assay performed in triplicate. Brackets indicate standard deviations. B and C. RNase protection assays of TRα (B) and Rev-erbα (C) expression in heads (0 days and one week) or cerebral cortex (2-18 weeks). Probes extend across exon 9A (TRα1) or the 5'ss of exon 6 (Rev-erbα) as indicated in Figure 1A, yielding protected bands of 156 or 137 nts for TRα1 or Rev-erbα, respectively. Control lanes include HEK 293 cell RNA which contains a low level of endogenous human RNA that cross-reacts with the opossum TRα probe (compare with a similar control with the opossum probe in Fig. 4D). Arrows indicate positions of the expected products. D. Electrophoretic analysis of realtime RT-PCR reactions illustrating absence of TRα2 expression in M. domestica. Analysis of reactions run on same cDNA sample (day 0 heads) with arrows indicating positions of amplification products for TRα1 and Rev-erbα mRNAs and positions of product expected from correctly spliced TRα2 mRNA. Three replica reactions for TRα2 show a variety of artifactual products but no detectable product of the expected size. E. Amplification of a synthetic TRα2 template based on homologous sequences from opossum. Threshold cycle values (CT) are shown for serial dilution of test DNA in the presence of a constant amount of cDNA from day 0 heads (which has no detectable TRα2). Efficiencies of 99.4% and 99.6% were calculated from two independent preparations of the gel-purified synthetic template.