Cell culture, transfection, RNA extraction and CFTR splicing pattern analysis
HeLa (human cervical carcinoma) cells were grown in DMEM (high glucose, GlutaMAXTM supplement, Gibco) with 10% foetal bovine serum (FBS, Gibco) at 37°C in a humidified incubator in the presence of 5% CO2. Cells were plated at a density of 4*105 cells in 6-well plates at 70% confluency 24 h before transfection. Transfection was performed using Lipofectamine 2000 (Invitrogen) with CFTR minigenes (500 ng) and Exon Specifics U1 (500 ng, for a total of max 1µg of DNA) and harvested 24 h later. After 24h transfection, RNAs were extracted using Maxwell® RSC simply RNA Cells Kit in Maxwell Automated RNA extractor (Promega). RT was performed using 1µg of RNA, and cDNA was used in PCR with Taq DNA Polymerase (New England BioLabs). Primers used to amplify CFTR minigenes were oligo-Forward (α 2,3): 5’-CAACTTCAAGCTCCTAAGCCACTGC-3’ and oligo-Reverse (Bra2) 5’-TAGGATCCGGTCACCAGGAAGTTGGTTAAATCA-3’. PCR products were separated on a 2% agarose gel running at 70mV for 30 minutes and the intensity of the bands were measured with ImageJ software (Rasband, 2012).