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Protocol to study the effects of mutations near splicing sites on pre-mRNA splicing
Daoyuan Xie1, Qiongling Peng2, Yu Tian1
1Laboratory of Translational Medicine Research, Deyang People's Hospital of Chengdu University of Traditional Chinese Medicine, No. 103 First Section of Taishanbei Road, Jingyang District, Deyang 618000, China.
Abstract:
Mutations at RNA splicing sites or regulatory elements can alter splicing efficiency or patterns, affecting RNA functionality and tissue-specific expression. Here, we present a protocol to study the impact of mutations near splicing sites on precursor mRNA (pre-mRNA) splicing. We describe steps for constructing plasmids by cloning the target gene into the pEGFP-N1 vector, performing site-directed mutagenesis, and transiently transfecting HEK293 cells. We then detail procedures for conducting mini-gene splicing assays to analyze splicing patterns influenced by mutations. For complete details on the use and execution of this protocol, please refer to Peng et al.1.
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