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.