RNA Analyses in Fibroblasts

Total RNA was extracted from cultured fibroblasts derived from skin biopsies from one affected individual (III:4) and the parents. In a total volume of 20 µl, first-strand cDNA was synthesized with 250 ng of total RNA using random primers and RevertAid First Strand cDNA Synthesis kit (Thermo Scientific). Polymerase chain reaction was performed using primers specific for exons 25 and 27 of NFASC. The PCR products were cut out from agarose gel and were purified using GeneJET Gel Extraction Kit (Thermo Scientific). The fragments were sequenced to validate the skipping of exon 26. Isoform specific expression levels of NFASC (NF186) were studied by quantitative real-time PCR (qPCR) on Quant-Studio 7 Flex Real-time PCR System (Applied Biosystems) using Power SYBRGreen (Life Technologies). All samples were run in triplicates, averaged, and normalized against total isoforms of NFASC. Mathematical model developed by Pfaffl (2001) was used for relative quantification of NFASC isoforms.