Cy0 - A new method in Real-Time PCR quantification

Why
To Adopt
C_{Y_s}

No need to estimate efficiency through assumption of equal efficiency between unknowns and standard curve

Efficiency Assumption Free

Reliability

Get quantification results also in very sub-optimal amplification condition

Extremely easy to implement in Real Time PCR instruments software

Integration

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The theorical basis of the invention lays in A new real-time PCR method to overcome significant quantitative inaccuracy due to slight amplification inhibition (Cy0), BMC Bioinformatics 2008, that still today register almost 200 downloads every month.

This result led us to start www.cy0method.org, in order to let subscribers test for free both our invention and SOD (Shape Outlier Detection), BMC Bioinformatics 2010, 11:186 (which we take into account to develop in the next months).
This site in less than 2 years has reached more than 30000 visits and is among first 5 results provided by Google by the search PCR quantification; moreover, a community of 150 subscribers from throughout the world uses our method with better results compared with the gold standard Ct.

Meanwhile the project started to get more and more attention among the international scientific community.
This led to relevant related publications both in important papers Validation of kinetics similarity in qPCR, T. Bar, M. Kubista and A. Tichopad, Nucleic Acids Research 40,4 2011, and books PCR Troubleshooting and Optimization: The Essential Guide, J. Gallup, Caister Academic Press, pp. 26 - 28, in which are highlighted the main features of the method compared with others.

Regarding the reliability and strongness of Cy0 (the theorical basis of the invention above mentioned) we delegate to Evaluation of qPCR curve analysis methods for reliable biomarker discovery: bias, resolution, precision, and implications, Jan M. Ruijter et al., Methods, Volume 58, Issue 1, sept. 2012.

As last, the several feedbacks collected and the analysis of hundreds of uploads performed by subscribers, have allowed us to hughly stress and improve the invention, in order to get more robust and reliable results in every (expecially sub-optimal) experimental condition in RT-PCR amplification processes.

Sothat, the invention CyS expands the features of Cy0:

Efficiency Assumption Free (i.e. no need to reviously extimate reaction efficiency)
hand free,

And introduces new ones:

more robust and reliable, allowing to get a quantification result in every experimental condition
estremely simple to adopt in PCR instruments software.

In other words, to adopt such a method would imply advantages from several points of view.

Working on the Cy0 method (about its reliability see this), through the analysis of hundreds raw data amplification runs collected through this site, every 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. This led us to realize a new pcr quantification method able to provide a valid result almost in every (suboptimal) amplification condition: the Cy_s.
Because of its reliability in every condition, we decided to relate the quantification data provided through this site to this new patent from now on.