RNA isolation, splicing analysis, and quantitative real-time PCR

Total RNA was extracted using TRI Reagent (Invitrogen), according to the manufacturer's instructions. One microgram of total RNA was reverse transcribed using random primers and the M-MLV Reverse Transcriptase (Invitrogen).

Endpoint PCR analysis of PISD, KRT15, and COL17A1 minigenes splicing was performed using the PISD ex3 dir and pCI rev, pY7 ex2 dir and KRT15 ex3 rev, alpha 2–3 dir and pFAN 904 rev sets of primers, respectively (Supplemental Table 1). PCR conditions were 94°C for 5′, 30 cycles at 94°C for 45″, 56°C for 45″, 72°C for 45″, and 72°C for 10′. Minigenes were transfected into cells along with 100 ng of pCF1 plasmid, which was used as control for normalization of transfection and reverse transcriptase efficiencies as previously reported (Pastor et al. 2011). PCR amplification of control pCF1 plasmid was performed under the same condition using CF ex1 248 dir and CF ex3 292 rev primers (Supplemental Table 1). PCR products were resolved by 2% agarose gel electrophoresis. The 1kb Plus DNA Ladder (Invitrogen) was used as molecular weight reference marker (M). The intensity of the bands was quantified with the ImageJ software. The percentage of EI isoform (%EI) is expressed as mean ± SD and calculated as the percentage of the isoform divided by the total abundance of the two isoforms.

Transcripts were quantified by using the iQ SYBR Green Supermix (Bio-Rad) in a CFX96 Real-Time PCR system (Bio-Rad). Primers used for cDNA amplification are listed in Supplemental Table 1. Each primer set used was tested by qRT-PCR using a 10-fold serial dilution of cDNA and water as nontemplate control in order to determine amplification efficiency, specificity, and the presence/absence of primer dimers (Supplemental Fig. S6). The amplification of a singular product with a uniform Tm was confirmed by the presence of a single peak in the melt peak curve. The amplification efficiency was determined from the linear slope of the standard curve: Only primers with a standard curve slope between −3.1 and −3.6 and an amplification efficiency comprised between 90% and 110% were considered suitable for further quantification. qRT-PCR cycling protocol was: 95°C for 3′, 40 cycles at 95°C for 15″, and 60°C for 1′. GAPDH and for keratinocytes samples RPL13A, RPLP0, and TBP (expressed as geometric mean) were amplified as internal controls.

For mature miRNA quantification, 1 µg of total RNA was reverse transcribed using the M-MLV Reverse Transcriptase in a reaction mixture containing a miRNA-specific stem–loop reverse transcription primer (Applied Biosystems). Quantification of mature miRNAs was performed using the TaqMan Universal Master Mix II and predesigned TaqMan probes (Applied Biosystems, Supplemental Table 1). qRT-PCR was conducted at 95°C for 10′, followed by 40 cycles of 95°C for 15″, and 60°C for 1′. RNU6B small nuclear RNA was amplified as an internal control. Results of SYBR Green and TaqMan assays were normalized to the internal control and relative quantification of each mRNA and miRNA was determined using the 2^−ΔΔCt method (Livak and Schmittgen 2001).