Transcript variation - measuring by RT-PCR - (Aug/18/2008 )
I am studying a gene that has multiple transcripts (I think 10-11 have been identified related to 10 known exon 1 regions - start codon part way in of exon 2). Some of these transcripts are not produced with specific SNPs.
I want a clean and quick way to determine which transcript is produced and in what ratio (compared to the main, functional transcripts). I will be comparing different tissues such as different brain regions.
At this stage, I am envisioning having to do 10 PCR reactions and comparing Ct values.
Has anyone else done this before that can offer some advice?
I think this is a risky thing if the difference between the expression levels of the different isoforms is small. I am studying a gene with only two different 1st exons, and the levels are very different from each other (1 is almost not expressed while the other one is comparable to GAPDH). The thing you should be careful about is the PCR efficiency. If you have only minor differences in your efficiencies, this may lead to skewed Ct values. I was once told by an expert in qRT-PCR that you should avoid comparing different amplicons. But if the differences are big, I think it can be feasible.
Hallo probably you can use multiple Real time PCR by using differently marked sonds, It is known that multiplex QPCR is hard to establish, but 2 sond should work, Design them in the overlaping region of the transcripts. It should reduce your work to 5 reaction mixture
or MLPA is way for detection of transcripts but do not offer quantity data
Thank you DPO and Baxa,
I agree that the differences between transcript levels may be small but if I multiplex, then maybe I can "ignore" the least transcribed forms and concentrate on the more highly transcribed forms.
Thanks for sharing :-)