Hi all,
I got a very simple query regarding minimum number of concentrations of 10-fold dilution standard curve. I know from reading around that, the unknown samples should fall between the range of standard curve for relative quantification. Am able to get only 5 points on standard curve for houskeeping gene (100, 10, 1, 0.1, and 0.01) and 4 points for gene of interest (100, 10, 1 and 0.1) with 98% efficiency for housekeeping and 96% for gene of interest. I will be using 10ng/ul (5ul of unknown cDNA in 25 ul of final rxn vol.) for each well, which makes up 50ng of unknown. Does anyone put light on this and help me to go on with the real time.

Thanks a lot

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to stay within your range, you can do more 1:5 or even 1:2 dilutions, if you want more data points for the curve?

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hello aimikins,
Thanks for your reply. As you know theoretically, there should be -3.3 difference between two 10 fold dilution concentrations (eg. 100ng-10ng=-3.3 of slope). What should be the difference between 1:5 dilution concentrations and 1:2?

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Hi,

Aimikins is right, you may use 1:5 or even 1:2 dilution to get more point of your standard curve.
If you use 1:10 dilution, interver of Ct will be 3.3. If you use dilution 1:5 or 1:2, Ct interver will be smaller, but slope remain the same as -3.3. Slope never chain.
Also remember, no matter how you dilute your standard or samples, the lowest detectable limit does not chain.

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hi,
thanks for your and aimikins help. Thats really appreciated for quick and experienced advice. I will try this and see how it goes.

cheers

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the cp-value difference depends on the efficiency. Try the following formula: log(dilution factor)/log(efficiency)=delta_cp

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thanks westenmax....

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