Cy0 is a new method in Real-time PCR analysis that does not require the assumption of equal efficiency between unknowns and standard curve. The method demonstrates that, in optimal amplification conditions, The Ct, second derivative (Cp), sigmoidal curve fitting method (SCF) and Cy0 methods methods are equally precise and accurate. However, when PCR efficiency was decreased, diluting amplification mix quantity or adding a biological inhibitor such as IgG, the SCF, Ct and Cp methods were markedly impaired while the Cy0 method gave significantly more accurate and precise quantification. For more details please visit cy0team.uniurb.it.