A quantitative RT-PCR protocol to adapt and quantify RBM20-dependent exon splicing of targets at the human locus

Gene splicing is a fine-tuned process orchestrated by splice factors including RNA-binding motif 20 (RBM20), and their mutations are linked to the development of cardiac diseases. Here, we provide a step-by-step protocol to transfer RBM20-dependent splicing from rat to human. This protocol describes a PCR-based approach to adapt and quantify RBM20-dependent exon-expression of human target genes. We detail the primer design, the use of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) for RNA isolation, followed by quantification of splicing products. For complete details on the use and execution of this profile, please refer to Streckfuss-Bömeke et al. (2017). [Display omitted] •Humanized primer usage to detect multiple splicing products by RT-PCR•Use of Sanger sequencing to annotate the exons included within a splicing product•Detailed description for primer design to quantify specific exon expression by qRT-PCR•Use of patient-specific iPSC-CM recapitulating RBM20-based dilated cardiomyopathy Gene splicing is a fine-tuned process orchestrated by splice factors including RNA-binding motif 20 (RBM20), and their mutations are linked to the development of cardiac diseases. Here, we provide a step-by-step protocol to transfer RBM20-dependent splicing from rat to human. This protocol describes a PCR-based approach to adapt and quantify RBM20-dependent exon-expression of human target genes. We detail the primer design, the use of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) for RNA isolation, followed by quantification of splicing products.