Hi all
i am new at designing new method for real time pcr and i want to detect some gene expression at klebsiella pneumoniae. i am using 5 ng cDNA template for reaction. My question is which concentration should i start for standart curves? 1/10 or 1/5 serial dilution i must do?
Hi Ibrahim,
I think that the concentrations of the standard curve must be set to the number of copies you expect to get in your samples.
Standard curves typically must have six points in serial dilutions of 1/10 for the Ct are sufficiently separated especially at low concentrations.
We work with curves ranging from 1 copy to 1000000 copies. But if you expect to find a number greater than 1 or 10 copies, need not reach these points on the curve but you must add points in the upper part of the curve to reach six.
I hope this information helps you.
I have found that the best first preliminary approach is to do an 18-point serial 1:2 dilution of a sample mixture, and test all targets of interest on that. Then, you can see the entire behavior of your qPCR over a great range. Once you see where the "sweet range" is, then you can decide what dilution factor to use to perfectly corral that particular range for standard curves. Inside that standard curve is a point to which you can dilute all experimental samples for individual target assessments. It is often the case that one finds that serial 1:10 dilutions are too aggressive for many moderately-expressed targets. Often a 5 or 6-point serial 1:3 or serial 1:4 standard curve works in most situations (after the "sweet range" has been identified up-front).
Thnx for answers.
@raquel i try 6 fold 1/10 dilution start from 40ng to 0.004ng but it is not effective because lower dilution didnt show peak. Also it cant be because of my low gene expression.
@jack i will try your comments. in fact i tried 1:10 and 1:2 dilutions 6 point but i cant see effective curve.
Best wishes for you
Hi Ibrahim, what do the first few dilutions look like then? Is the efficiency good for the first few dilutions (i.e. ~3.3 cycles difference per 10 fold dilution)
At roughly what Ct are you seeing the 40 ng dilution? If you know the sequence of the cDNA and the cDNA is pure, you can calculate the copy number that 40 ng represents. If it is a mix of cDNAs then is there another gene that you can reliably detect and dilutes well?
If there is a large gap in Ct after a few dilutions, it is possible that too much cDNA is stuck to the tube/pipette tip as well. This discussion has a post about that.
https://www.researchgate.net/post/How_can_I_analyse_this_absolute_quant_qPCR_standard_curve
@jeremy my first dilution series 1/10 starting 40ng isnt very efficient (R2= 0.78) but today i try 5 point 1/4 dilution starting with 80 ng to 0.3125 ng and qpcr effiency is 0.992. Also no unspecific peak when i am doing melting curve analyse.
So you have solved the problem? What did you differently? Starting from 80 or 40 ng should not make too much difference ~ 1 cycle at .992 efficiency.
Of course you can test some different concentration as an optimization expriment and after that you use the best one for major expriment and your own samples according to suitable standard curves.
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