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All the ten exons verified were alternatively spliced in at least one of the sampled tissues (Additional file 3 Figure S2), and, in the cases where it could be quantified from the brightness of the corresponding bands in the gel, the trends of exon inclusion levels were in agreement with the microarray analysis.
Using Sanger sequencing of cDNA based PCR products, all three candidates were indeed verified as being alternatively spliced in P. patens protonema and gametophore tissue, respectively.
The experimentally verified existence of the alternatively spliced exon 1A is in agreement with an identified cattle 5'-EST sequence (GenBank: EG705801).
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Expression of alternatively spliced transcripts was verified by RT-PCR and DNA sequencing; primer sequences are listed in Table S1.
ERα binding to alternatively spliced genes was verified by chromatin immunoprecipitation assay.
Several examples of alternatively spliced transcripts were verified by qPCR, some of which showed significantly different expression patterns.
For ELF3 gene that has not been studied, the presence of alternatively spliced RNA variants was verified by direct sequencing of RT-PCR products.
Alternatively, the indirect method can be verified theoretically using Simpack.
Alternatively, the activity of LDL-R was verified using radiolabelled LDL-[H]-Cho.
ERα binding to select genes that were alternatively spliced in response to E2 was verified by ChIP followed by real time PCR (ChIP-qPCR) as described previously [ 48].
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Justyna Jupowicz-Kozak
CEO of Professional Science Editing for Scientists @ prosciediting.com