RT-PCR of Minigenes
After 24 h incubation, total RNA was extracted using TRIzol reagent (Invitrogen, USA) and used for RT-PCR to confirm the splicing patterns. First-strand cDNA was synthesized from 2 μg of total RNA by random-primed reverse transcription with Superscript II Reverse Transcriptase (Invitrogen Corporation, Carlsbad, CA). To evaluate the pattern of transcripts from the transfected minigenes, the following vector-specific primers were used for RT-PCR amplification: a forward primer SD6 (5’-TCTGAGTCACCTGGACAACC-3’) and a reverse primer SA2 (5’-ATCTCAGTGGTATTTGTGAGC-3’).
The PCR amplification reaction was performed as follows: in 50 μl volume, 2 μl of cDNA, 10 μL of 5 × PrimerSTAR Buffer (TaKaRa, Japan), 1 μM of each primer, 0.8 μlM dNTPs, and 0.5 μL PrimerSTAR HS DNA Polymerase (TaKaRa, Japan) in a 9700 (Applied Biosystem, Foster City, CA, USA) thermal cycler. Thermal conditions were 33 cycles of 94 °C for 30 seconds, 58 °C for 30 seconds, and 72 °C for 90 seconds, and followed by a final elongation step at 72 °C for 10 minutes. The PCR products were separated by electrophoresis on a 1.5% agarose gel, and each band signal was quantified by software Image J. The target DNA bands were purified using a Gel Extraction Kit (CWBIO, China), and then all transcripts were analyzed by sequencing.
Statistical analysis
The percentage of exon exclusion (%) was calculated as (lower band / [lower band + upper band]) × 100. Statistical analysis was performed using SPSS software. The results were analyzed using the two-tailed Student’s t-test or One-way ANOVA test by GraphPad Prism (Version 6.02, GraphPad Software, USA). Error bars represent SEM (n=3). A P value < 0.05 was considered statistically significant.