32p γ atp and pnk

Total RNA was extracted from cells using TRI reagent (Ambion) according to manufacturer’s protocol. The RNA was digested with 2U RNase-free DNase I (Ambion) for 30 min at 37 °C before phenol extraction and precipitation. Reverse transcription was performed using 500 ng RNA with random primers and RevertAid reverse transcriptase (Fermentas) according manufacturer’s protocol. For radioactive PCR analysis of alternative splicing, primer sets were designed across the constitutive exons. The list of primer sequences is shown in Additional file 8. To radioactively label the primers, 2.5 µM of each primer were incubated with ³²P-γ-ATP and PNK (Fermentas) at 37 °C for 30 min before purifying through a G6 column (Biorad). PCR reaction was performed with DreamTaq polymerase (Fermentas) using 1 µl of hot primers and 1 µl of cDNA for 25 cycles at 60 °C annealing temperature. The 2x RNA Loading Dye (Ambion) were added to the PCR products, and were denatured at 95 °C before resolving on the 8% denaturing polyacrylamide gel. Results were quantified with the Typhoon Imager (GE healthcare) and ImageJ software.

Total RNA was extracted from cells using TRI reagent (Ambion) according to manufacturer’s protocol. The RNA was digested with 2U RNase-free DNase I (Ambion) for 30 min at 37°C before phenol extraction and precipitation. Reverse transcription was performed using 500 ng RNA with random primers and RevertAid reverse transcriptase (Fermentas) according to manufacturer’s protocol. For radioactive PCR analysis of alternative splicing, primer sets were designed across the constitutive exons. The list of primer sequences is shown in Additional file 8. To radioactively label the primers, 2.5 μM of each primer were incubated with ³²P-γ-ATP and PNK (Fermentas) at 37°C for 30 min before purifying through a G6 column (Biorad). PCR reaction was performed with DreamTaq polymerase (Fermentas) using 1 μl of hot primers and 1 μl of cDNA for 25 cycles at 60°C annealing temperature. The 2x RNA Loading Dye (Ambion) were added to the PCR products, and were denatured at 95°C before resolving on the 8% denaturing polyacrylamide gel. Results were quantified with the Typhoon Imager (GE healthcare) and Image J software.