Figure 4

Splicing of chimeric TRα minigenes. A. Schematic showing intron-exon structure of TRα minigene. Light shading indicates the TRα2 3'UTR. Vertical brackets indicate a 3.2 kb deletion in intron 9. Heavy horizontal line represents the exon 9A RNase protection probe (panel D). Arrows represent primers used for PCR (panels B and C) as described in Methods. Primer positions for TRα2 mRNA are indicated separately for rat (solid arrows) and opossum (dashed arrows); those for TRα1 are located at nearly identical positions. The composition of chimeric minigenes is indicated by horizontal bars aligned with structure at top: dark and light shading represents opossum and rat sequences, respectively. B, C. Electrophoretic analysis of conventional (B) or realtime (C) RT-PCR of RNA from transfected HEK 293 cells using primers indicated in Panel A. Large arrows indicate PCR products from correctly spliced TRα1 or TRα2 mRNAs. Dotted arrows indicate spliced products obtained from cryptic splicing within exon 10. Larger products seen with TRα2 primers from R-O, O-O and O/RpA represent readthrough of unspliced or partially spliced RNAs, low levels of contaminating DNA (Panel B) or non-specific products (Panel C). ΔC_T values shown in Panel C represent differences observed between threshold cycles (C_T) for TRα1 and TRα2 (ΔC_T = C_T^TRα1 - C_T^TRα2) averaged over three replica assays from each of three independent experiments. D. Analysis of minigene splicing by RNase protection assays using probes for exon 9A. E. Summary of results in panels B, C and D. Plus signs represent reactions where correct splicing of TRα2 was detected following sequencing of conventional PCR products. % splicing was calculated from ΔC_T values (see Methods) with standard deviations given in parentheses. Asterisks represent possible false positives from non-specific RT-PCR products or high background in RNase protection assays.