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defining ratio of alternative spliced gene with qPCR

alternative splicing qPCR

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14 replies to this topic

#1 Anpu

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Posted 05 September 2013 - 05:36 AM

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



#2 bob1

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Posted 05 September 2013 - 02:28 PM

The calibrator would be the normal form of the gene.



#3 Anpu

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Posted 05 September 2013 - 11:27 PM

That is how i thought after reading hundreds of articles.

 

So in my case every tissue has its own calibrator? 

 

thank you for help.



#4 doxorubicin

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Posted 06 September 2013 - 12:56 AM

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



#5 Anpu

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Posted 06 September 2013 - 01:55 AM

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"?



#6 doxorubicin

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Posted 06 September 2013 - 02:09 AM

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.



#7 Anpu

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Posted 06 September 2013 - 02:10 AM

Thank you for the help.



#8 Anpu

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Posted 09 September 2013 - 12:37 AM

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



#9 bob1

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Posted 11 September 2013 - 03:33 PM

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



#10 Anpu

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Posted 11 September 2013 - 11:16 PM

thanks!

 

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



#11 doxorubicin

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Posted 03 October 2013 - 06:07 AM

 

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

 

hello again smile.png

 

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 smile.png

 

thank you for the help.

 

regards

 

Sorry, I just saw this question directed to me.  Assuming you're using a SYBR Green protocol...To make the standard curve do the following:  Run the qPCR exactly as you plan to do for the final experiment (same RT from the same cells with the same primers and the same master mix). When the reaction is finished, upon the PCR plate, collect the completed reaction, and purify the product with a kit (Qiaquick PCR purification or equivalent). Use a nanodrop to determine the exact concentration of your purified product.  Then prepare a dilution series 1:10, 1:100: 1:1000, 1:10,000, 1:100,000, 1:1,000,000 of the purified product in TE buffer.  People usually add some sheared salmon sperm DNA for stability.  Then test the dilution series using your qPCR protocol and see if it is linear with an efficiency <100%.

 

It is very likely that you will contaminate your pipetteman with purified PCR product.  It might be better to use one from another lab to set up the standard curve.

 

Mark



#12 Anpu

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Posted 03 October 2013 - 06:47 AM

Hi Mark.

 

Thank you for the reply. I ordered gBlocks from IDT. Just the other day the sails rep was here and we ere talking about it and he told me that this can be done with gBlocks. More and more people are using them and it great! They are not expensive, so my boss agreed :)

 

Regards and again tank you for the help!

 

Andrej



#13 atashia

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Posted 05 October 2013 - 09:35 PM

sdzfzfx



#14 Anpu

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Posted 24 October 2013 - 07:04 AM

hi everyone!

 

I have another question about the absolute quantification.

 

Now that i have analyzed some samples i have noticed that some quantity numbers are surprising.

 

I have done one quantification with SYBR green (one plate) and another with TaqMan probes (other plate) no other way. 

 

Do I have to change the treshold to the same value at bot runs or there is no problem if they are different? For TaqMan is lover that SYBR green!

 

Thanks for the help.

 

Best regards.

 

Andrej



#15 Anpu

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Posted 15 July 2014 - 01:32 AM

Hi again.

 

i stumbled upon another problem. With the absolute quantification where CTs in triplicates have just 0,1 CT standard deviation but when it is calculated to absolute molecule quantity the standard deviation with that can be greater than 10% between triplicates.

 

Does anyone have any Idea why? Ok i know the pipetting is probably the main problem but how do I explain that with statistics and in article.

 

Thanks for answers.

 

Regards, Andrej







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