Quotes from the internet: qPCR and efficiency curves

To me, anytime I see people demanding that you have at least a 6 log dynamic range to work with, I know immediately they really haven’t performed that much qPCR with low abundance targets, and they also blindly believe that purified amplicon, plasmid or other differently purified target source will magically amplify with exactly the same efficacy as it amplifies within the biological sample extracts we ultimately assess by qPCR.

-JMG (in the Biotechniques forum)

 

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  1. #1 by Jo Vandesompele, UGent professor, Biogazelle CEO on April 7, 2012 - 19:11

    While there is some truth in these statements, we have extensively tested (> 300 assays) and verified the equivalence between the PCR efficiency determined by dilution series of cDNA made from reference RNA or made from synthetic templates. We typically use 60 bp single stranded oligonucleotide templates. I agree that PCR efficiency in real biological samples may be different than using (high quality) reference RNA or synthetic templates, but this can be tested. For assay validation, reference RNA or synthetic templates are adequate.

  2. #2 by Alejandro Montenegro-Montero on April 7, 2012 - 21:28

    Thanks for the comment. It surely can be tested!

    People tell me that what they usually use as template for testing efficiencies is a plasmid where the amplicon has been cloned. I’ve only heard of just a couple of people use reference RNA. I wouldn’t think (and I agree with you there) that there would be much of a difference when using (purchased) reference RNA anyway (compared to using RNA obtained in the lab), provided your sample is relatively clean, but sounds like a great idea (when available, i.e for *some* model organisms).

    I use a pool of cDNA derived from the same organism, processed in the same way as my samples.

    In any case, I still agree with the ‘in-between the lines’ message of the quote, which is that it’s wrong *to assume* that efficiencies will be the same. As you mention, testing is the way to know.

    We typically aim for amplicons between 75-120 bp when designing qPCR primers. Is there a particular reason for using 60bp templates?

    • #3 by Jo Vandesompele, UGent professor, Biogazelle CEO on April 7, 2012 - 22:03

      For real amplicons, we also aim for that range. However, for the synthetic template, we order 60 bp ss unmodified oligo as this is the longest oligo that can typically be synthesized at small scale and low cost without extra purification. Again we tested that the efficiencies are equivalent for short and long amplicons. It is all about the primers!

  3. #4 by Ajith on April 1, 2013 - 18:26

    what is the relation between sample volume and Ct value?? 1ng/uL DNA sample gives 25Ct when 4uL of the template is used in a 10uL reaction and when 1.2uL of the same is used in a 6uL reactio n Ct remained the same. How is it possible??

    Thanks in advance

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