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PCR exons 5 to 7

PCR exons 5 to 7. 300 bp. ins14. wt. del47+ins14. 200 bp. del47. skipping of exon 6. M. 2. 1. 3. 4. Supplemental figure (E)F-2: Results of RNA analysis

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PCR exons 5 to 7

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  1. PCR exons 5 to 7 300 bp ins14 wt del47+ins14 200 bp del47 skipping of exon 6 M 2 1 3 4 Supplemental figure (E)F-2:Results of RNA analysis Results of a RT-PCR with the primers 5F and 7R separated on a 4% agarose gel. M = marker, 100bp ladder (MBI Fermentas); 1 = muscle cDNA, patient; 2 = muscle cDNA, control; 3 = cDNA of HEK 293 cells transfected with CHRNE IVS5-16G>A containing minigene; 4 = cDNA of HEK 293 cells transfected with wild-type CHRNE minigene. In wild-type muscle and in cells transfected with the wild-type minigene (lanes 2 and 4) the wild-type splice product of 251 bp is generated, accompanied by a weak band of the del47 alternative splice product (204 bp). Cells transfected with the mutated minigene (lane 3) generate an ins14 transcript (265 bp) along with a del47+ins14 variant (218bp). In the patient’s muscle, all four transcript variants can be detected (lane 1). The transfected cells (lanes 3 and 4) produce a high amount of exon 6 skipped transcript (150 bp); this transcript type can hardly be detected in muscle tissue (lanes 1 and 2).

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