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QPcr for alternatively spliced products - (Aug/22/2007 )

Hello everyone,
I was wondering if there was a way to do Qpcr with alternatively spliced products. The primers I am using create two bands, and I want to quantitate the difference between them. Is this at all feasible?

-tara123-

you mean in the same tube with the same primers?

I don't think that this is possible quantitatively because if you have one more abundant splice product it will "munch" the reagents (primers, dNTPs,...) and therefore could also influence the kinetics for the other pcr product.
IMHO you have to do it in separate tubes with primers that can distinguish between the splice variants.

-Ned Land-

Yes in the same tube, the primers I have test for both genes at the same time. ie say gene a = 3, 4, 5 and gene b = 3, 5 (alternatively spliced). The real problem is, I transfect a "mini-gene" which recapitulates the endogenous splicing of the cell. For that I created original sequence outside of the minigene so I can test for the mini-gene only and not get interference with endogenous products. So, I can't think of anyway to engineer the primers so that they only detect one band, but without endogenous interference. Does this make any sense?
Tara

QUOTE (Ned Land @ Aug 22 2007, 12:09 PM)
you mean in the same tube with the same primers?

I don't think that this is possible quantitatively because if you have one more abundant splice product it will "munch" the reagents (primers, dNTPs,...) and therefore could also influence the kinetics for the other pcr product.
IMHO you have to do it in separate tubes with primers that can distinguish between the splice variants.

-tara123-

Just a guess - can you use two different taqman probes, one in the common region fo your gene (which shows you the 100% amount amplified) and one only present in the variant. You would have one signal for the whole gene and one for the variant (given that you use different probes) and you should be able to calculate from there huh.gif

-kr├╝melmonster-

You may design one taqman probe in the junction 3-5 and other in the junction 3-4.
with the firs probe yoy quantify the alternative splicing and with the other the constitutive splicing, you can the 3-5 in one color, ie FAM and the other in other colour.

-davidjsanz-