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molecular biology

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molecular biology

Postby irdabn » Tue Apr 17, 2007 7:45 am

my cDNA has 9 exons, with the 2nd exon being spliced alternatively

i want to detect both the wild type containing all the 9 exons and the mutant with 2nd exon deletion both and quantitate them. how do i do this in real time pcr. any one can suggest me whether i can use lux primers
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Postby blcr11 » Wed Apr 18, 2007 2:32 pm

I think LUX methods might work. Certainly, you should be able to make the labelled products, but whether you can quantitatively distinguish the two types of transcripts with LUX or not I’m not sure. If you run the pcr products on a gel, you should be able to see the different sizes, but then LUX won’t be useful and you’ll have to use something else (gel scanning?) for quantitation.

I don’t know if this will work or not. What if you used a probe for, say, Exon 9—almost any exon(s) but Exon 2, and then a separate reaction with a probe just for Exon 2. I guess there’s nothing to stop you from making probes to each exon separately. The signals for all the exons but 2 should be the same and include all transcipts, so you should be able to get a good average. The signal from Exon 2 should be only the full transcript (the transcipt containing all exons). Then the signal for the ∆2 transcripts would be the difference between the average of all the other wells and the signal in the Exon 2 alone wells. Does that make any sense?
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Postby blcr11 » Wed Apr 18, 2007 2:36 pm

I was assuming that the alternate splicing would be present in the same cell. If that's not the case, then why wouldn't an Exon-2-specific vs any-other-Exon-probe give the quantitation you need--or am I missing something?
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Postby blcr11 » Wed Apr 18, 2007 3:46 pm

Perhaps it would be possible to use probes with different emission maxima (colors) for the two classes of transcripts allowing independent quantitation that way. Might want to take a look at the specs on the Invitrogen website to see if that is a possibilty.
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