A double-reporter splicing assay for determining splicing efficiency in mammalian cells.
Changes in alternative splicing patterns can result from both inherited and acquired defects, and they are increasingly recognized as causes of human diseases. Hence, improvements in the understanding of alternative splicing regulation may provide opportunities for restoring productive patterns of splicing. The identification of factors (such as proteins, nucleic acids or small molecules) that modulate the splicing pattern would be facilitated by systems with which many samples can be screened. The absence of reliable systems prompted us to develop an assay system based on dual enzymatic activities. Two distinct signals derived from spliced and unspliced RNA are measured, providing the basis for a robust, rapid and convenient assay for investigating splicing. This protocol describes how to use this system; the time required for lysing the cells and recording enzymatic activity is about 2 hours.
Nasim
Md Talat
MT
Department of Medical and Molecular Genetics, King's College London, Guy's Hospital, London SE1 9RT, UK. talat.nasim@genetics.kcl.ac.uk
Eperon
Ian C
IC
D017398:Alternative Splicing / Q000235:genetics* / Q000502:physiology*; D000818:Animals; D002460:Cell Line; D004847:Epithelial Cells / Q000378:metabolism*; D017930:Genes, Reporter / Q000235:genetics*; D005821:Genetic Techniques; D006801:Humans; D008322:Mammals; D022821:RNA Splice Sites / Q000235:genetics
D022821:RNA Splice Sites