qPCR - urgent help (Apr/07/2009 )

Hello mates,

I am performing a cotransfection reaction for an effector and a reporter genes, and i want to test the effect of the effector gene on the expression of the reporter one, but the problem is that i am i have the same vector for both of them (pcDNA3.1).
previously, when i performed one construct transfections, i have used neomycin gene (in the pcDNA3.1 plasmid) for normalization, i dont know how i will normalize the qPCR results now for the expression of the reporter plasmid since i have neo in the effector and the reporter plasmid.
best regrads

Sorry, I am a little bit confused here...........
Why you're doing qPCR? What do you want do detect?
Are you supposed to do reporter assay such as lacZ or luciferase assay?

To start the story from the beginning, the reporter plasmid consists of a minigene construct (3 exons and a small part of there introns).
One of these exons (exon 2) is alternatively spliced, and i want to measure the expression levels of each isoform of the minigene( the full-length and the delta exon2).
For that, in this experiment i have the effector plasmid, that is differen splicing enhancers and silencer binding proteins.
After all, i want tto perform a cotransfection experiment to check the effect of each (splicing enhancers or silencers binding protein) on the expression levels of both isoforms by qPCR.
I dont have any reporter assay, i am performing a syber green qPCR, using gene specific primers.

Functional Screens on Apr 7 2009, 04:17 PM said: