Application Guide - QIAxcel Advanced

QIAxcel Advanced Application Guide 10/2016 25 The extracted pre-mRNAs were subjected to RT-PCR and sub- sequently analyzed using the QIAxcel system. Samples of low DNA concentration were analyzed using Method OL400. Samples were injected at 8 kV for 20 s, and separation was performed at 6 kV for 400 s. Alignment marker, with fragments of 15 bp and 3 kb, was injected at 4 kV and 20 s and run simultaneously with the samples. QX DNA size marker, with fragments ranging from 50–800 bp, was used for size and concentration estimation. High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) was performed as previously described (6) using an antibody specific to Tra2β (7). Results and Discussion As expected, analyses on the QIAxcel system demonstrated very high splicing activation by full length Tra2β, but also significant percentage splicing inclusion (PSI) activation by the Tra2β∆RRM–GFP protein (Figure 1). These results indicate that for some exons, Tra2β can act as a coactivator as well as a splicing activator. Interestingly, Tra2β∆RS1 seems to behave as a potent splicing repressor. This indicates that the endogenous Tra2β∆RS1 isoform acts a splicing repressor and/or that the RS1 domain plays a central role in splicing activation. Conclusions • The QIAxcel system is a valuable tool for revealing exon splicing patterns. • Analyses of the splicing patterns using the QIAxcel system were both qualitative and quantitative. • The QIAxcel system can help to identify the roles of other splicing proteins as activators, coactivators, or repressors. A B Figure 1. Splicing of Nasp-T M3+M4 (mutant) exon. A. Percentage splicing inclusion (PSI) of a panel of exons identified through HITS-CLIP in response to GFP and Tra2β-GFP fusion proteins. Data represent at least 3 biological replicates, and the error bars are shown as standard errors. B. Representative image from RT-PCR analysis on the QIAxcel system. Probability (p) values were calculated using an independent two-sample T-test between the PSI levels for cells cotransfected with GFP and each of the different Tra2β-GFP constructs (p≤0.05, p≤0.01) (adapted from reference 5). 100 80 60 40 20 0 Region coverage GFP F P 2 β M S 1 Tra2β Tra2β Tra2βΔRRM Tra2βΔRRM Tra2βΔRS1 230 – 188 – PSI Alt exon 100 80 60 40 20 0 Region coverage GFP G F P T r a 2 β T r a 2 β Δ R R M T r a 2 β Δ R S 1 Tra2β Tra2β Tra2βΔRRM Tra2βΔRRM Tra2βΔRS1 230 – 188 – PSI Alt exon

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