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defining ratio of alternative spliced gene with qPCR - (Sep/05/2013 )

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Hi everyone. 

 

I'm new here so I apologize if I am writing in wrong forum.

 

My question is:

 

I have tissue samples of different organs and different animals.

 

I'm looking for the ratio of a gene and its alternatively spliced form (isoform).

 

Does anyone have any idea with which method to calculate the ratio. I have Ct values that I've gotten them with qPCR. I am using the endogenous controls.

 

If I go with the method 2^(-delta delat Ct) I need a calibrator and I don't know if I can use any of the samples because I don't know what is the state 0 (the isoform is present everywhere in every tissue).

 

in  short:

 

- want to calculate ratio between two forms of same gene (wild type and its isoform).

- I have tissue samples (can't gen cell cultures or anything)

- working with qPCR

 

Thank you all for help.

 

Regards.

 

Andrej

-Anpu-

The calibrator would be the normal form of the gene.

-bob1-

That is how i thought after reading hundreds of articles.

 

So in my case every tissue has its own calibrator? 

 

thank you for help.

-Anpu-

A better approach would be to generate a standard curve for each PCR product and determine absolute quantities (ng) of each product. 

-doxorubicin-

A better approach would be to generate a standard curve for each PCR product and determine absolute quantities (ng) of each product. 

 

And calculate ratios out of absolute quantities?

 

But isn't relative quantification better and more "precise"?

-Anpu-

Yes, you would calculate the ratios from the absolute quantities....it is the most precise you can be.  For the delta-delta Ct method you make the assumption that your efficiency is 100% for each PCR, which is not true, so it is less precise.

-doxorubicin-

Thank you for the help.

-Anpu-

A better approach would be to generate a standard curve for each PCR product and determine absolute quantities (ng) of each product. 

 

hello again :)

 

doxorubicin: I am thinking about absolute quantification and I'm thinking about the standard curve.

 

What is the best way to make standard curve? I was thinking about synthesizing DNA of my gene and tha go from there :)

 

thank you for the help.

 

regards

-Anpu-

You could do the synthesis, but it is probably easier cheaper to have a plasmid containing the gene of interest.

-bob1-

thanks!

 

I'll make synthesis by IDT gBlocks (if anyone already used them). Its cheap.

-Anpu-
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