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PCR Efficiency


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#1 JonBio

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Posted 17 November 2009 - 01:24 PM

Good Afternoon,

We are trying to calculate PCR efficiencies for several genes that we are working with. We are using SYBR Green with a Stratagene qPCR machine. We made 6 serial dilutions of cDNA. All of our calculated efficiencies were around 80% with R2 values around 0.997. I know the acceptable efficiency is around 90-110%, but will this be a problem if we are comparing genes with similar efficiencies using the delta delta Ct method? Does anyone have any ideas on where to start to increase the efficiency? Thanks for your help.

Jon

#2 BioMiha

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Posted 18 November 2009 - 05:32 AM

Good Afternoon,

We are trying to calculate PCR efficiencies for several genes that we are working with. We are using SYBR Green with a Stratagene qPCR machine. We made 6 serial dilutions of cDNA. All of our calculated efficiencies were around 80% with R2 values around 0.997. I know the acceptable efficiency is around 90-110%, but will this be a problem if we are comparing genes with similar efficiencies using the delta delta Ct method? Does anyone have any ideas on where to start to increase the efficiency? Thanks for your help.

Jon


Do you run a dissociation curve and run your products on an agarose gel after the PCR? If not, perhaps your reaction is not as specific as you might think. Maybe there are some primer dimers or non-specific products.
Other things to consider is adding some betaine or DMSO if your product is forming secondary structures. Also adding a bit of proofreading polymerase might help, if the one in the mix is halting at certain points.
Just my 2 cents.
Also in my oppinion the R^2 values are misleading, because in a log lin plot there will be much less scatter than in a lin lin plot therefore greatly inprooving the R^2.

Cheers,
Miha

#3 JonBio

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Posted 19 November 2009 - 10:28 AM

Thanks for the response Miha. I guess I am a little confused as to what affects the efficiency of PCR. We are extracting RNA from skeletal muscle using Trizol. We resuspend the RNA in TE buffer and treat with Turbo DNase. After quantifying the RNA we are using 40 micrograms to proceed with our RT reaction. We typically dilute the cDNA 1:10 for qPCR, and use 4 microliters of this dilution. We usually get around 80% efficiency for these conditions. We have found that when we dilute the cDNA further (1:40 or 1:80), our efficiency increases to 90-110%, and the Ct value is around 30. Could this be a solution or would it be better to increase the concentrations of other reagents instead of reducing the amount of cDNA? I guess it could be a number of different things in the master mix. But do primer concentrations relative to cDNA typically have the largest effect on efficiency? We have run our products on a agarose gel after PCR and get one band at the expected size.




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