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qPCR


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#1 thegene

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Posted 07 April 2009 - 05:17 AM

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

#2 Functional Screens

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Posted 07 April 2009 - 04:17 PM

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?

#3 thegene

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Posted 08 April 2009 - 12:01 AM

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.




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?






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