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Different cDNA volumes for target and reference genes in qPCR?

Different cDNA volumes for target and reference genes in qPCR? - Molecular Biology Techniques

Different cDNA volumes for target and reference genes in qPCR? - Molecular Biology Forum. Includes forums for common molecular biology techniques.


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Old 06-25-2013, 03:05 PM
Pipette Filler
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Default Different cDNA volumes for target and reference genes in qPCR?



Hi

Am I able to use 2ul cDNA template for qPCR of my target gene samples and use 1ul for my reference gene, or do they always have to have exactly the same DNA volumes?

Thanks!
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Old 06-26-2013, 04:41 PM
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Default Re: Different cDNA volumes for target and reference genes in qPCR?

The problem is the final volume of the reaction. Commonly one prepares the master mix for ALL samples, including problem and controls. Then you add the proper volume to each tube (is the same volume for each one). In the end, after adding DNA (either genomic or complementary) template, the final volume should be the same in all tubes. Therefore, if you change the volume of template DNA you add it will ultimately change the final concentration of the master mix reagents (Mg+, dNTPs, etc.).

Since qPCR is specially sensitive, minor changes between reactions might affect the reproducibility of your results. In other words, you might have trouble infering if your results are caused by the conditions of the samples or by discrepancies between reaction tubes.

In summary, yes, it always has to have the same DNA volumes. Regarding solutions, you can mix your reference DNA in one volume of diluent (nuclease free water, TE buffer or whichever you use) so you can add 2 uL like for your target samples.

Good luck
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Old 06-27-2013, 01:00 PM
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Default Re: Different cDNA volumes for target and reference genes in qPCR?

Thanks very much for your reply. I was planning on adding nuclease free water to the reference gene samples to make sure the total reaction volume is the same for everything so I'm glad you agree that this is ok.

So am I right in thinking that it doesn't matter if the concentration of DNA is different between my samples and the reference gene samples as it is relative quantification so the difference between the two for the different treatments is the important thing rather than the actual ct values that are generated?

Just want to make sure I get it right before I go pipetting a 96 well plate!

Thanks!
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cdna , genes , qpcr , reference , target , volumes


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