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

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

The calibrator would be the normal form of the gene.

That is how i thought after reading hundreds of articles.

 

So in my case every tissue has its own calibrator? 

 

thank you for help.

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

doxorubicin on Fri Sep 6 08:56:04 2013 said:

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"?

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.

Thank you for the help.

doxorubicin on Fri Sep 6 08:56:04 2013 said:

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

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

thanks!

 

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