how to see splicing.. - what do you think about what I planned? (Mar/14/2007 )

Hi can you give me your opinion?!

I want to see if I can get a new variant of a transcript.
The group I work with, has already characterize a protein (VMP) that plays a role during the stress response in pancreatitis, so we have the sequence and its location in the genome (of rat) and so forth.
And in a NB it can be seen not only the band expected for the known transcript but also a second band, and even a third one in some tissues; so based on that I want to try something to see if I can have more information of the second band.
I did some bioinformatic analysis of the gene sequence and determinated its exons and introns and so far I have this conclusions:

VMP has 12 exons and its length is of 1.9kbp

The unknown NB band (a possible second mRNA) has around 2.7kpb

Then using an exon prediction program (genscan) I analized the VMP genome sequence. It predicted many exons included 11 of the 12 that form VMP (it ignores an exon that is before the one that has the ATG).
And in this configuration:
exons 2,3,4 are predicted one after the other
between 4 and 5 there are a lot of other predictions
the same happended between 5 and 6
6 and 7 are one after the other too
7 and 8 have other predictions between them
8,9,10 are one after the other too
there is a gap between 10 and 11
and finally 11 and 12 are detected one after the other.

So my idea is.....to isolate the RNA, do RT PCR with a poly T primer and then do PCR with a Fw primer from the second exon of VMP ('cos we have a primer for the ATG site at the lab) to see if I have more than one amplicon, only if I'm lucky and they share that exon.....and other issue that can bother me is if the RT and the Taq can't read the whole sequence (I don't know if 1.9 and 2.7kb are too long for the enzimes).
And then based on what results I get from this, maybe I could also do some reactions with primers to amplified the gaps between the exons I mentioned above.

Other idea I imagined is that maybe I could do something like the protection nuclease assay, but instead of hibridizing genome DNA and mRNA I could hibridize the cDNA of VMP with the unknown mRNA (only if I can get it from the first PCR), supposing that the exons in common are going to hibridized and the rest is degraded with the enzime.

They are just ideas, can you give me your opinion??

thank you

biotech.

The ideas sound OK, but a bit complicated. Could you isolate the second RNA from the gel, or purify by another means, then do the RT step, blunt end and clone into a plasmid? If so, you can sequence from the plasmid (so you don't need to know the sequence of the 5'end of the transcript).
Potential problems: I don't know how well a DNA/RNA hybrid will clone, if at all.

How do you know that the exon before the ATG is not part of the gene? Does it not have a promoter sequence associated? Does it not have its own ATG? If the 'second' exon is the one with the ATG, why is it not the first exon?

If you want to see what exons are in the longer transcript, can you do a Northern using single exons as the probe? That should tell you which exons you can use as primers for your RT-PCR idea.