Transfection and RT-PCR analysis
The minigene plasmid transfected into HEK293T cells with lipofectamine
3000 reagent (Thermo Fisher Scientific, USA) based on manufacturer’s
instruction. After 48h, collected the cells and then performed RT-PCR to
evaluate the mRNA splicing. Briefly, total RNA was extracted using
TRIzol reagent (Sigma-Aldrich) and then reverse transcribed to cDNA by
reverse transcription kit (Vazyme) according to the instruction. PCR was
performed to amplify the fragment containing the mutant site. All
primers were listed below: forward: 5’- ATGGTGGTGTTCGGGTATGA-3’,
reverse: 5’-AGCGTCTGAACAG GAAGTTTG-3’ (for lymphocytes, exon 1-3);
forward: 5’-GAAGCGCCCAAGGTTTTCAA-3’, reverse:
5’-ACTATGCCAATCAGTGAGCTTCT-3’ (for lymphocytes, exon 2-4); dSD-F:
5’-TCTGAGTC ACCTGGACAACC-3’, dSA-R: 5’-ATCTCAGTGGTATTTGTGAGC-3’ (for
HEK293T cells). Agarose gel electrophoresis (BioFroxx, Germany) was used
to separate the segment of PCR products. Then the sequence of each DNA
product was determined by sanger sequence.