4.4. RNA Extraction and RT-PCR

Total RNA was harvested from transfected Type I SMA fibroblasts 48 h after treatment with AOs using Trizol reagent, according to the manufacturer’s protocol (Invitrogen, Waltham, MA, USA). The RNA solution was then stored at −20 °C until use. Primers used to assess expression of hnRNPA1, SMN and other transcripts are listed in the supplementary Table S2 [36,37,38,39,40,41,42,43]. Approximately 200 ng of total RNA from each extraction was used as templates for single-step RT-PCR (Invitrogen, Waltham, MA, USA). Amplicons from the hnRNPA1 and SMN RT-PCRs were fractionated on 2% agarose gels and images were captured after staining with ethidium bromide. Product intensity was measured on an ImageQuant LAS 4010 (GE Healthcare Life Sciences, Chicago, IL, USA) and densitometric analysis was determined using image J software [44]. Percentage of SMN2 exon 7 inclusion is calculated by FL-SMN signal divided by the summation of density values of FL-SMN and SMNΔ7. The percentage of hnRNPA1 inclusion was determined as the density of exon 7b inclusion divided by the density values of the hnRNPA1 band and the hnRNPA1b band.