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[Trof]

Hi,
I have a rather complicated experiment.

Two cell lines, a control one (number 5) and one with the insertion I'm testing (number 2). I wanna do a elongation test by a real-time pcr using a cell nuclei. I have the nuclei and in vitro transcribe them, then measure the amount of the transcript on the exon2 and on the exon3. We need to compare it, because we expect the in vitro transcription to halt in the insertion before exon three, so there will be more copies of exon2 then exon3 in the tested cell line (and same copies of both in the control cell line, where the transcription reaches the end).
To complicate it even more, I have untreated nuclei that contain small original amount of mRNA present there at the time of the isolation, which is something like a base level, that i need to substract from the values of the in vitro transcribed ones.

Question is how to calculate this.

So far I thought about geting Cts from all the samples, control (#5) and tested (#2) in both transcribed and untranscribed for both exons and a dilution curve to get efficiencies for each exon.

Then, can I appoint it to a standard Pfaffl equation?

E(exon2)^deltaCt exon2 (#5 - #2)
-------------------------------------------- = relative ratio
E(exon3)^deltaCt exon3 (#5 - #2)

Because I want to have a ratio of exon2/exon3 for #2 using exon2/exon3 values of #5 as a reference, that means equal to 1. I also need to correct for the different efficiencies of each exons, that's why I don't use delta-delta Ct.
Is my calculation right?

Then there goes the problem with the Ct values of the untranscribed samples, how to substract them from the Cts of transcribed samples? Or can I ommit them, since they really doesn't matter? (I don't care how much there is exon2 or exon3 transcript copies, only their ratio is important and that stays the same in the untranscribed samples as well).

Any help appreciated.