A quantitative RT-PCR protocol to adapt and quantify RBM20-dependent exon splicing of targets at the human locus

Figures

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  Figure 1. Blast of ldb3 primers for rat against the human LDB3 locus (A) The ldb3 forward primer for rat (Guo et al., 2012) was blasted against the human LDB3 locus. The primer shows 3 mismatches compared to the sequence of the human exon 4. (B) The ldb3 reverse primer for rat (from Guo et al., 2012) was blasted against the human LDB3 locus. The primer shows 2 mismatches compared to the sequence of the human exon 7. The color code in A and B is related to the human LDB3 locus. (C) Schematic summary of localization of primers for ldb3 in rat (from Guo et al., 2012) in ENSRNOT0000083623.1 and the localization on the human locus in LDB3 in ENST00000623056.4.

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  Figure 2. DNA bands after PCR amplification of LDB3 using primer “LDB3 exon 4–7” for semi-quantitative RT-PCR (see key resources table) on an agarose gel The two bands were excised from the gel, and the DNA bands were purified for Sanger sequencing. Sequencing results in combination with Clustalomega-based blasting demonstrated exons 4, 5, 6, 7 in the upper band and exons 4, 6, 7 in the lower band.

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  Figure 3. Relative Expression of LDB3-exon 5 Differential mis-splicing for exon 5 in LDB3 in RBM20_S635AiPSC-CM. Every dot represents one differentiation experiment. Data is shown as bar graph with standard error (control).

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  Figure 4. Example tracks of good and bad sequencing results Left: Example of a good Sanger sequencing result. The peaks of the respective nucleotides are clearly separated. Right: Example of a bad Sanger sequencing result. Nucleotide peaks are super imposed, and no clear sequence can be retrieved.