In Vitro Splicing Assays

In vitro splicing assays represent an important technique to analyze, in detail, the basic splicing mechanisms for a pre-mRNA of interest. The cell-free assays, under defined conditions, have many advantages for characterizing the cis-acting elements and trans-acting factors responsible for the regulation of alternative splicing. Radiolabeled transcripts of minigenes of interest are spliced in HeLa cell nuclear extracts under various conditions, and the splicing products analyzed directly using denaturing polyacrylamide gel electrophoresis, or detected indirectly using RT-PCR. Here, a standard method of in vitro splicing is described that has been used successfully for many years in the authors' laboratories.