Protocol for High-Resolution Mapping of Splicing Products and Isoforms by RT-PCR Using Fluorescently Labeled Primers

Abstract

Summary: We describe an RT-PCR protocol that allows high-resolution mapping of splicing products and isoforms using fluorescently labeled primers. Each species contains one fluorescent group allowing a direct comparison of the different isoforms despite size differences. A custom-size ladder enables the precise determination of cDNA lengths and discrimination of isoforms differing by less than five nucleotides on polyacrylamide gels. This protocol also allows the detection of products from in vitro splicing reactions, circumventing the need to use radiolabeled transcripts.For complete details on the use and execution of this protocol, please refer to Gabunilas and Chanfreau (2016).