Evaluation of absolute quantitation by nonlinear regression in probe-based real-time PCR

Background: In real-time PCR data analysis, the cycle threshold (CT) method is currently the gold standard. This method is based on an assumption of equal PCR efficiency in all reactions, and precision may suffer if this condition is not met. Nonlinear regression analysis (NLR) or curve fitting has therefore been suggested as an alternative to the cycle threshold method for absolute quantitation. The advantages of NLR are that the individual sample efficiency is simulated by the model and that absolute quantitation is possible without a standard curve, releasing reaction wells for unknown samples. However, the calculation method has not been evaluated systematically and has not previously been applied to a TaqMan platform. Aim: To develop and evaluate an automated NLR algorithm capable of generating batch production regression analysis. Results: Total RNA samples extracted from human gastric mucosa were reverse transcribed and analysed for TNFA, IL18 and ACTB by TaqMan real-time PCR. ...

• Type: Journal
• Year: 2006
• Where: BMCBI
• Authors: Rasmus Goll, Trine Olsen, Guanglin Cui, Jon Florholmen
• Keywords: Absolute Quantitation, Analysis, BMCBI 2006, Regression Analysis