Cys: beyond the fitting.

Kind reader,

 after one year it was about time for some news.
Since the main work inspiring this site (Guescini et al. [2008]), we got many feedbacks and tens of uploads which allowed us to expand the view on the result already achieved:
Cy0 is still the first method in Real-time PCR analysis that does not require the assumption of equal efficiency between unknowns and standard curve.
As shown, the Cy0 method and the Gold Standard Ct (and others) were equally precise and accurate in optimal amplification conditions, while, when PCR efficiency was decreases, diluting amplification mix quantity or adding a biological inhibitor such as IgG, the Cy0 method gave significantly more accurate and precise quantification.
Moreover, the analysis of the data collected through this site, has given us the cue to find a path leading to a quantification method able to provide a valid result almost in every (suboptimal) amplification condition.
In this sense, every global model we dealed with, has shown fitting problems in particular shaped amplification curves, i.e. when the flex point is too close to the X-axis, or as well, dealing with Taqman chemistry curves, either Sybr Green.
All this, suggested to follow another approach which lead us to realize a new method: Cys.
Because of its reliability in every condition, we decided to relate the quantification data provided from now on to this new procedure.
At last, we would like to take this opportunity, to thank the hundred and more subscribers that with their interest have allowed our project to achieve this new result.
Sincerely, The Cy0 Team

Related Publications

Feedbacks
  • "I have been evaluating the Cy0 in a qPCR assay to determine copy number variation in genomic DNA, using TAQMAN (FAM/VIC) chemistry. I estimated the Cy0, using the R qpcR library, from a 5 parameter log-logistic model fitted to the deltaRn values (automatic baseline, ABI 7300). In over 450 samples, I haven\'t identified any apparent sample quality problems, but have observed occasional, sporadic FAM automatic baselining problems. Importantly, the Cy0 method is relatively insenstive (but not completely invariant) to these baselining issues in terms of CN assignment, whereas the recommended Ct (0.2 threshold) gives rubbish results. In all other samples, the Ct and Cy0 have given identical, discrete, high confidence CN calls."
    Sue Lester, Australia 25/03/2011
  • Dear Cy0-team,
    thanks for the chance to use your method for PCR-quantification. I came across your method, when looking for other ways f or QPCR than using Linreg, realtime PCR-miner, DART and other programs, which claim to give efficiency values for correction. Now, in my hands all these methods give widely differing results and all have much greater variance for replicates than the good old delta Ct-method. Now, even though I do not understand all the maths in your procedure, it performs at least as well (or even better) as the delta Ct-method..." Thanks agains, and I will continue to submit data.
    Sincerely, Matthias
    Dr. Matthias Kirsch - Univ. Freiburg,
    Institut für Anatomie & Zellbiologie
    Abteilung Neuroanatomie
    Zentrum f. Neurowissenschaften, ZfN.
    Freiburg, Germany 2/3/2011
  • Dear Cy0-team,
    I noticed thar Cy0 method were more precise than the Ct method, a 5 % difference in some results (i.e: RSD (%) of 8,8 to Cy0 against 14,4 to Ct). But this was only true to sample with sufficient DNA sample.
    I had some assays with low DNA samples that Ct got better results. I figure it was the lack of Data to estimate Fmax, that distort the Cy0 values.
    My experiments were design with a Full factorial Design (2^3), and I noticed that i had a greater resolution using Cy0 results. I work with a system to extract Adenovirus, and used PCRq to analyse my results. Thank you for your help. I hope this info were of any help.
    João Vitor Dutra Molino
    Faculdade de Ciências Farmacêuticas - USP
    Cidade Universitária São Paulo - Brazil 10/3/2012
  • Dear Cy0 team,
    “Thanks for the quick results. I would like to have to following comments on the results.
    1. By using the Cy values for plotting the standard curve, the R square values are generally increased (e.g. 0.9985->0.9995, 0.9972->0.9975, 0.9931->0.9996, 0.9995->0.9996 and 0.9978->0.9995; Ct value from ABI SDS 2.4-> Cy).
    2. The reading between the technical replicates are closer, i.e. lower SD.”
    Raymond WK CHEUNG
    Department of Medicine and Therapeutics
    Li Ka Shing Medical Sciences Building
    Prince of Wales Hospital
    Shatin, N.T. - Hong Kong S.A.R.