Fig. 3

Analysis of the impact on RNA splicing of APC c.721G > A by using a cell-based minigene splicing assay. a Structure of pCAS2-APC.ex7 minigene used in the assay. The bent arrow indicates the CMV promoter, boxes represent exons, lines in between indicate introns, and arrows below the exons represent primers used in RT-PCR reactions. The WT and c.721G > A minigenes were generated by inserting a genomic fragment containing the exon of interest and flanking intronic sequences into the intron of pCAS2, as described under Materials and Methods. b Analysis of the splicing pattern of pCAS2-APC.ex7 WT and c.721G > A minigenes. The two constructs were introduced into HeLa cells and the minigenes’ transcripts were analyzed by RT-PCR 24 h post-transfection. The image shows the results of a representative experiment in which the RT-PCR products were separated on a 2.5% agarose gel stained with EtBr and visualized by exposure to ultraviolet light. M, 100 bp DNA ladder (New England Biolabs). c Quantification of splicing events observed in the minigene splicing assay. The relative levels of exon inclusion indicated under the gel are based on RT-PCR experiments equivalent to those shown in B but performed with a fluorescent forward primer and then separated on an automated sequencer under denaturing conditions. Quantification results were obtained by using the GeneMapper v5.0 software (Applied Biosystems) and correspond to the average of two independent fluorescent-RT-PCR experiments. d Representative fluorescent RT-PCR experiment. The panel shows superposed peaks corresponding to the WT and mutant products (in blue and red, respectively), as indicated