Search for CFTR exon 2 skipping by reverse-transcriptase PCR amplification of mRNA from monocytes. (A) RT-PCR design: mRNA from monocytes was analyzed by RT-PCR using primers designed to amplify fragments of different length, depending on the presence or absence of exon 2, to better define if the genetic variant 186-8T/C had caused alternative splicing in this subject. Total RNA was purified and reverse-transcribed with random primers. PCR was then carried out for 35 cycles (30” at 94°C, 30” at 60°C, 30” at 72°C) using the forward primer, Ex 1–4 Forward 5’-AAAGGCCAGCGTTGTCT-3’ (black arrow) or W/O exon 2 Forward primer 5’-ACTTTTTTTCAGAGAATGGGATAGAG-3’ (gray arrow), and the reverse primer, Exon 1–4 5’-GCGATAGAGCGTTCCTC-3’ (black arrow). The exon-spanning primer “W/O exon 2 Forward primer” is localized between the exons 1 and 3 coding sequence, and thus works properly only in the absence of exon 2. Amplified fragment lengths using Exon1-4 Forward and Reverse primers (black arrows) were expected to be 339 bp. Therefore, in the case of exon 2 skipping, we would expect a shorter PCR-product (228 bp). With the exon-spanning Forward primer (W/O Exon 2; grey arrow) we expected an amplified product of a length of 207 bp. (B) PCR-amplification products: We compared the PCR-amplified products obtained from the patient (186–8 T/C), with a healthy subject heterozygous for the common F508del mutation, and with a healthy subject (no mutations) as internal control. The amplification product obtained from the subject carrying the 186-8T/C variant, using Exon 1–4 Forw/Rev primers, was undistinguishable from controls, moreover, using the exon2-spanning primer no amplification products were observed in the samples from any of the subjects (Negative Controls-NC). As control we amplified a portion (290 bp) of the housekeeping gene actin; this PCR was then carried out for 25 cycle.