I'm interested to know if anyone had tried competitive FISH on mRNA sequence of 60% similar isoforms. What if these isoform did not have decent nucleotides gaps to provide specific probes? Is it possible to use competitive FISH? I know that the method is quite common in genomic comparative, thus I'm interested if the same concept could be use in studying protein expression, in specific, the regulation of mRNA in translating 2 or more isoforms.
Fareed,
I just saw your question, although it was posted a while back.
You're welcome to conctact me with details on your mRNAs and I'll see how we can help you.
You can also find details on how we do singlemolcule RNA FISH here:
www.biosearchtech.com/stellaris
Hans
Hi Hans,
Thanks for the reply. I had already used the Stellaris FISH probe from biosearch and it was very good. We manage to identify one of the isoform mRNA. We tried with the other isoform but no luck. When both mRNA was evaluated with multiple alignment method, they has 60% difference of sequence. However, we did not obtain any reading from the second isoform. either the cells did not have the second isoform mRNA or our flow cytometer unable to detect the signals. We do test both FITC and CalFluor for signal and no problem. Inthe end,we confirmed one of the isoform and manage to proceed other experiment with the results. Attach is one of the paper we publish and hope you can provide some insight on the results.
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