Qualitative and quantitative evaluation of alternative splicing products using the digital droplet PCR

Background The CFTR gene encodes for a chlorine-transporting protein essential for the correct hydration of epithelium in many organs and tissues. The malfunction of CFTR protein causes Cystic Fibrosis, a genetic disease with an incidence of about 1 out of 3000 births. To date, more than 2000 CFTR gene mutations are known and many of them alter the correct splicing of its mRNA. The qualitative evaluation of the effect of a splicing genetic variant is a simple procedure that by an RT-PCR followed by an electrophoretic separation can reveal the alternative splicing products. Conversely, making a quantitative and accurate evaluation of splicing products, in order to evaluate the percentage of proper residual splicing, requires complicated and expensive procedures. Materials and methods We evaluated the use of digital droplet PCR (ddPCR) with EVAGreen technology to quantify the amount of exon skipping due to splicing mutations. In particular, we used total RNA extracted from nasal cells brushed directly from subjects bearing different intron 9 polyT variants, including the 5T-12TG allele. The total RNA was retro-transcribed and amplified by ddPCR using a single primer pair to amplify both PCR products, 305 bp (correct splicing) and 122 bp (Exon 10 skipped). Results Using a single primer pair it was possible to determine different percentage of exon 10 skipping depending on the poly T tract size, as reported in literature. In particular, we obtained about 50% and 95% correct splicing from 5T/7T and 7T/7T subjects. Conclusions These data, even if preliminary, strongly suggest that it is possible to accurately quantify the percentage of correct residual splicing with a simple pair of primers, without the use of special probes. This methodology could be helpful especially in the analysis of composite heterozygous, in which splicing mutations are associated with other severe mutations and it is necessary to know the exact contribution of the splicing variant to the phenotype.