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Correcting for efficiencies and differences among replicate Ct values

RT PCR Absolute quantification

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#1 GradMicro

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Posted 02 August 2013 - 03:52 PM

Hi, I am using two real time PCRs , one a genus specific and another a species specific one to profile the quantities of a bacterial genus and a species in clinical samples.

Despite repeated checking with standard curves, the efficiencies of the two PCRs are different and i need to find a method to normalize this. I am not sure if I can apply the efficiency correction for standard vs sample in this case or are there any other formula that i can use.

 

 

Secondly, is there a CLSI or other guidelines that gives recommendation on how to proceed where there are differences in the CT values of the replicate of samples, I use triplicates in all my samples plus standards and ge differences of about 0.25 - 0.0001 in the ct values among the replicates. Not so sure as to what exactyl to do in these instances, take the average, drop outlier etc. Different papers seem to take their own method but I would like to know a general consensus for this.

 

Thanks a lot.



#2 traveler9907

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Posted 29 August 2013 - 07:28 PM

Standard dev of 0.167 or less is optimal for tech replicates. Use the Pfaffl method for gene expression. Be sure you did a full dynamic range for the standard curves, otherwise, the error rate in efficiency is huge! At least 5 logs, 10-fold is best. Check this out for help. Www.bio-rad.com/supermixes_tutorial. Found it useful.

#3 GradMicro

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Posted 29 August 2013 - 08:05 PM

Thanks a lot. My aim is bacterial load quantification, not gene expression. Thanks for the link too.







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