Genetools image analysis software 4

RNeasy Fibrous Tissue kits (QIAgen, Manchester, UK) were used in RNA extraction. Muscle tissue was homogenized in the lysis buffer provided with the kit at 20 Hz for 2 min, twice, using a TissueLyser II (QIAgen, Manchester, UK). The total RNA was then extracted following the manufacturer’s instructions. RNA quantification was performed on a ND-1000 NanoDrop spectrophotometer (Thermo Scientific, Leicestershire, UK). Five-hundred nanograms of extracted RNA were reverse transcribed by GeneScript RT-PCR kit (GeneSys Ltd, Surrey, UK) and resulting cDNA samples were then amplified through RT-PCRs. Two microlitres of RT-PCR products were used as templates in subsequent semi-nested (dystrophin exon skipping) or nested (myostatin exon skipping) PCRs. The final PCR products were then loaded onto 2% agarose gels. HyperLadder IV (Bioline, London, UK) was used as a size standard. Densitometric analysis of gel electrophoresis results was performed using GeneTools Image Analysis software 4.02 (Syngene, Cambridge, UK). The efficiency of dystrophin or myostatin exon skipping was evaluated as a percentage of the density of skipped products against the total density of unskipped and skipped products. RT-PCRs were performed in duplicate. Details of PCR programs and sequence of primers (MWG, Ebersberg, Germany) are available on request.