new splicing variants identification - (Aug/10/2009 )

Hi,
I would like to identify splicing variants of my gene of interest. Which methodical approach would you recommend? I tried EST sequences, but there are not any. Nevertheless, from my experiments I know that there are at least several protein variants and I have "by accident" identified a new slicing variant of mRNA. I am thinking about multiplex RT-PCR, but I am not sure...
Anyway - how would you distinguish posttransciptional and posttranslational splicing and is there an approach how to assign the mRNA variants identified by RT-PCR and sequencing to the bands identified by western blot?
Thanks for your ideas

torte on Aug 10 2009, 01:49 AM said:

You can do any or many of the following.

1. Blast against sequence databases (ESTs etc).

2. Do RT-PCRs using different primers (in different exons) and different tissues/cell lines (splice variants may vary according to sources).

3. If splice variants are known in a different organism for the same gene, look for similar variants in your organism.

4. IP down your protein, run on an SDS-PAGE, cut out the bands in your protein size, and submit for sequencing. You may find splicing variants as well as post-translational modification variants.

5. Screen cDNA libraries from various tissues and cancer cell lines.

6. Interrogate various alternative splicing databases, especially in the species of your interest.

Hi,

regarding the splice variant search I have a question too.

I have a gen with 5 exons but only the last exon contains the cds. I found in WesternB my protein perfectly in P5 mice but when the mice are about P60 there is a complet shift. The original protein disappears and a protein half the size of the original appears. So obviously one would say there is no post modifications which would increase the size of the protein, so only a new splice variant in the 5th exon might be possible.

What do you think how could I detect on a mRNA level this smaller protein, which I guess is really a version of my first protein? Would you use a specific pattern of Primer an if so how would you design it.


Any Idea?

Thx

Andreas