Quantitative real time pcr

Total RNA from cells and tissues was extracted with Trizol Reagent (Thermo Fisher Scientific) following the manufacturer’s protocol. SuperScriptIV reverse transcriptase (ThermoFisher Scientific) was used for cDNA synthesis. The cDNA was then used for quantitative real-time PCR (BioRad), using SYBR Green qPCR mastermix reagent (ThermoFisher Scientific). The following primers were used:
Deup1 (deleted exons)
For: 5’- GCC AGA TGT AGA CAT TTC TTG GCA TGG −3’
Rev: 5’- CCC ACC TCC TGG CCT TT −3’
Deup1 (exons 10–12):
For: 5’- TAC GTC TTC CAG AGC CAG C −3’
Rev: 5’- CAG GAA GTG CTG TGC AGC −3’
Deup1 (exons 9–10):
For: 5’- GAA TTA AGC AAG GCT GTG GAC T −3’
Rev: 5’- CTC TGG AAG ACG TAT GCC CC −3’
Cep63 (exons 6–8):
For: 5’- ATC AGA CCT ACA GTT CTG CC −3’
Rev: 5’- CTG ACT TAG AAT CTC CTT ATG CTC −3’
Cep63 (exons 13–14):
For: 5’- GCA GGA GGA ATT AAG CAG ACT −3’
Rev: 5’- CTG TCG GAA TTC CTC TAT TTT TCC AG −3’
GAPDH:
For: 5’- AAT GTG TCC GTC GTG GAT CTG A −3’
Rev: 5’- GAT GCC TGC TTC ACC ACC TTC T −3’.
All samples were normalized to GAPDH (ΔCt). The relative fold change in mRNA expression as quantified using the 2^−ΔΔCt method comparing experimental samples to controls. All data was then normalized to the average value for the control samples.