qPCR

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Nelsson
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qPCR

Post by Nelsson » Mon Mar 30, 2009 1:52 pm

Hi! I'm trying to understand the mechanism of qPCR and how the results are interpreted. In qPCR a dye is used, which binds to dsDNA, and then the machine measures the fluorescense. The fluorescense is plotted against cycle number and the resulting graph shows which of the tests that contained a higher concentration of DNA, and thus takes fewer cycles to reach the Ct and plateau phase. Am I right so far? So, let's say I want to investigate the expression of a number of different genes. Since mRNA is converted to cDNA by reverse transcriptase, and a highly expressed gene is transcribing more mRNA than a down- regulated gene, I reckon that an up- regulated gene will have more starting material(cDNA) than a down regulated gene, and thus require fewer cycles to reach the plateau phase. But since this is a quantitative approach, I want to have some numbers to work with. I need some help to understand some of the answers. I've calculated the efficiency values for every test, which tells me how efficient the reaction has been. Is a higher/lower efficiency value somewhat related to the amount of templat DNA? I've also calculated the expression ratios using the Pfaffl method. I don't know, however, how to interpret these results. Is there some easy way you can describe what these numbers mean? high vs low expression ratio? can you have negative values, if so what does that mean?

I would really appreciate some help! Thanks :)

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canalon
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Post by canalon » Mon Mar 30, 2009 3:45 pm

I am not sure I understand your problems, but here is a tutorial I used when I first started using Real time PCR, and it helped me a lot: http://pathmicro.med.sc.edu/pcr/realtime-home.htm
I hope it will help you too.
Patrick

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)

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