Poster #091
ASO-Mediated RBFOX2 Loss Modulates RNA Exon Inclusion
Mentors: Katherine Rothamel, PhD, Lab member: Maya Steever
To investigate how varying RNA-binding protein (RBP) expression levels affect gene expression and mRNA splicing, we designed three different antisense oligonucleotides (ASOs), which are ASO1, ASO2, and ASO3, targeting the splicing factor RBFOX2. HEK293T (human kidney) cells were transfected with three different ASOs and a control ASO to knock down RBFOX2 expression. We first assessed the knockdown efficiency of each ASO using immunoblotting, comparing the expression of RBFOX2 protein levels with those of a housekeeping control. To evaluate the impact of altering RBFOX2 expression on splicing, we examined known RBFOX2-regulated targets using quantitative PCR (qPCR) with primers flanking cassette exons that undergo alternative splicing upon RBFOX2 loss. Splicing alterations were assessed via gel electrophoresis. Of the three ASOs tested, ASO3 achieved the most effective knockdown of RBFOX2. We are currently optimizing qPCR primers to more precisely quantify differential splicing events in response to RBFOX2 depletion.