RNA preparation and RT-PCR
Total RNA from human kidney (ThermoFisher AM7976) was used together with
total RNA from whole blood samples and urine-derived renal epithelial
cells (hUREC) isolated using RNeasy mini kit (Qiagen) according to the
manufacturer’s instructions and quantified using a NanoDrop 2000
spectrophotometer. 0.75µg RNA was reverse-transcribed using an oligo-dT
primer and SuperScript III Reverse Transcriptase (Thermo Fisher
Scientific). The resulting cDNA was used for PCR with a GoTaq® DNA
Polymerase (Promega) and a CC2D2A gene- specific primer pairs (5-
TGAGAGACACTGGCTGGGAT -3 and 5- AGGCACTGACGATTTGGAAAC -3) to identify
basal skipping of exon 30. Primers were designed using NCBI Primer-BLAST
(https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi).
Amplification of HPRT1 housekeeping gene cDNA was performed
alongside. Product electrophoresis was performed on a 2% agarose gel.