I have doubt or whether or not mRNA is maturing normally and I want to examine one or two transcripts, can I distinguish both by PCR with apropriate primers?
I think a lot depends on exactly what you're using as your definition of 'mature', and correspondingly, what stage you think your maturation is getting stalled at. If you could provide a little more information, it would be helpful.
I mean, the answer is ostensibly "yes", but it's a very qualified yes, and may or may not apply to your specific scenario.
Well, it depends. You could certainly look for the mature mRNA, assuming you have a tissue/condition that it is normally highly expressed. Normal cDNA preparation would find it. But it's really hard to find "immature" RNA molecules, especially if they are subject to the Nonsense-Mediated Decay pathway. mRNA without a cap degrades quickly in the nucleus. mRNA without a poly-A tail is never exported to the cytoplasm.
What step do you think isn't proceeding normally and why?
with the term immature I think you mean mRNA with introns, because polyA tail directly added by polymeraseII at the final stage. Cap is also initially added.
For this you can isolate nuclei which is enriched by immature mRNAs, than you can make cDNA synthesis by using random hexamer primers and then you can make qRT-PCR that one of the primers contains an intronic region. That way you can quantify immature mRNAs.
In agreement with Enes Yağız Akdaş
cDNA can be synthesized from total RNA, by using random hexamer primers, and quantitative real-time PCR should be done by two different primer pairs considering the intron region (i.e., exclude intron for mature mRNAs and for the immature mRNAs consider intron sequence as a part of the primer sequence).
Good luck
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