Revisiting the sigmoidal curve fitting applied to quantitative real-time PCR data.

摘要: Amplification of a cDNA product by quantitative polymerase chain reaction (qPCR) gives rise to fluorescence sigmoidal curves from which absolute or relative target gene content the sample is inferred. Besides comparative C(t) methods that require construction reference standard curve, other focus on analysis sole amplification curve have been proposed more recently. Among them, so-called fitting (SCF) method rests an empirical model experimental data points, leading prediction efficiency and calculation initial copy number in sample. The implicit assumption this may describe quantitatively even portion where signal hidden noise band. theoretical basis SCF was revisited here for defining class might be relevant. Applying six well-characterized different PCR assays illustrated possible pitfalls biased estimates and, thus,