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Northern blot with mRNA - (Feb/17/2010 )

Hi, there!

I would like to detect some transcripts spliced forms of my gene, so I was thinking in detecting them by northern blot, but using mRNA instead of total RNA. I was considering two possibilities:
1.- Extraction of total RNA from my cells, and then try to purificate mRNA from the total RNA by using poli-T columns or something like that.
2.- Extraction of total RNA from my cells, RT-PCR with oligo dT and finally PCR to amplify my gene of interest (I suppose i will have to amplify different regions of my gene to obtain every transcript which could be present).

After any of these two possibilities, I would continue loading the result of 1 or 2 into my agarose gel.

What do you think it's the better option? Do you know some other method?

Thanks in advance.


Option 1 is a northern, option 2 is not it is a PCR.


bob1 on Feb 18 2010, 12:04 AM said:

Option 1 is a northern, option 2 is not it is a PCR.

Maybe I failed to explain in an easy way my question... I suppose both options are called Northern, because of they are hibrydation procedures involving RNA. My question was about the previous modification of nucleic acid that I'm trying to detect later with a radiolabeled probe... All my fears come because I suppose that when I introduce total RNA into the gel, I'll detect both immature and mature RNA's, although I'm only interested in detecting mature RNA's.

Thanks for your time.