As far as I can see the literature is using total RNA for RNA-Seq analysis. This is fine if you're doing gene expression level. But there are numerous studies regarding alternative splicing events.
My question is, isn't using total RNA, which includes hnRNA from nucleus, inaccurate to report alternative splicing events?
Correct me if I am wrong but using polyA selected RNA out of total RNA is not sufficient filter out unspliced transcripts.
I'm sure there are labs doing cytoplasmic mRNA RNA-Seq as we speak, I'm wondering if it's okay to use RNA-Seq from total RNA to discover novel splice variants, relative expression levels of splice variants, etc.
In other (biological) words, doesn't polyadenylation occur before splicing is complete?
thanks,
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