DNA copy number and calculating % gmo

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Monique
Monique's picture
DNA copy number and calculating % gmo

Hello everyone!
I am new in q real time pcr and I have problem.
I don't know to calculate a % of gmo based on copy numbers.
I do relative quantification, for example rapeseed, I have one standard curve (based on copy numbers 300000,30000,3000,300,30) for reference genes and another for gmo modification.
I do not know what to do with my quantities.
Or If I have value in copies how to convert it in %  of gmo?
 

Ivan Delgado
Ivan Delgado's picture
 

 
Hi Monique,

What you are trying to do is a little tricky, but here is what I suggest. You already have qPCR assays for your GMO modification and for an internal standard (reference gene). Now what you need is a DNA sample that you know does not have any GMO in it (reference sample), and if possible a second reference sample that is 100% GMO. I would even go as far as mixing your 0% GMP sample with your 100% GMO sample in different quantities so that instead of running a standard curve based on copy numbers, I would run a standard curve based on GMO %, for example: 0%, 1%, 5%, 10%, 20%, 50% 75%, 100% GMO. 

Hope this helps.

Monique
Monique's picture
Thank you for your answer.

Thank you for your answer.
I have already tried to work with % gm (mixing) but I don't know what is going wrong.
Purpose of my experiment is to get % gmo from unknown samples.
After DNA isolation, samples are going into RT PCR. I use ISO21570 protocol for quantification.
My standards (when I use CRM %# don't show logic Ct values, for example I have higher Ct for higher concentrations.#).
So I have a lot of problems...If you have some experiences with good protocols, Ill
appreciate every help.
I use Stratagene Mx3000P RT-PCR.

Ivan Delgado
Ivan Delgado's picture
 

 

Hi Monique,

Unfortunately what you are trying to do is highly dependent on the assay you have and the RNA/DNA standards you are using. If you do not have a good assay, or very well characterized template standards, then it is going to be very tricky to get the assay to work successfully. 

Unfortunately I am not aware of an assay you could do for this as most of these assays are designed specifically for the given GMO being tested. 

Sincerely,

Ivan

Monique
Monique's picture
Thank you for reply!

Thank you for reply!
Best regards,
Monique