Minigene splicing assay of the c.1515+1G>A variant in NPNT.

(A) The pET01 construct used in this study. The sequences containing the c.1515+1G>A variant in NPNT intron 10 (A allele) or those that did not (G allele) were subcloned into the multiple cloning site of the pET01 vector. The arrows under the 3’ and 5’ exons indicate the primer pair used in RT-PCR after transfection. The primer sequences are provided in S2-1 Table in S1 File. (B) RT-PCR analysis using HeLa cells transfected with the wild-type, mutant, or empty (mock) pET01 vector. The RT-PCR products were separated in a 3% agarose gel electrophoresis and were stained with ethidium bromide. (C) Sequencing chromatograms confirmed the exon 10 was totally skipped in the mutant RT-PCR product.