How to amplify intronic antisense transcript? - (Sep/07/2006 )

hi everybody,

I'm trying to find a natural antisense intronic RNA. I have read that some antisense RNAs don't have poly-A tail, so I did RT from total RNA using random hexamers instead of oligo-dT. Before knowing the orientation of the possible transcript, I just wanted to know if actually the transcript exists. Using a couple of intronic oligos in the PCR, I obtain a band of the correct size (400 bp). There is no band in the control RT done without reverse transcriptase, so there's no genomic DNA contamination.

The question is: is it possible that I'm amplifying unprocessed "sense" RNA, and not an independent transcript different from the "sense" mRNA?

Thanks a lot.

-chico-

QUOTE (chico @ Sep 7 2006, 05:31 PM)

I wouldn't feel like excluding the possibility... I guess you are isolating total RNA, therefore you are isolating unprocessed and mature RNA... Stated beforehand that it could give you a contamination anyway, I would probably try to isolate only cytoplasmic RNA. I know that Invitrogen has a reagent to do that and I found it as good as Trizol in terms of yeald and purity of RNA when I used it

-dnafactory-

OK, thank you very much!

Anyway, do you have an idea of how long does an unprocessed RNA stays unprocessed? I mean, when I do normal PCR with oligos from different exons (let's say with a 400 bp intron between them), I never obtain a band corresponding to the unprocessed RNA (if the control RT- is clean).

Thanks again

-chico-

Try polyA tailed RT-PCR described in following paper and then sequencing your PCR products, otherwise, you should know the exact size of your products
using 72C for extension if your products is >200bp

Biotechniques. 2005 Oct;39(4):519-25.

-rye-