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

BMC Bioinformatics
Rasmus GollJon Florholmen

Abstract

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. To develop and evaluate an automated NLR algorithm capable of generating batch production regression analysis. Total RNA samples extracted from human gastric mucosa were reverse transcribed and analysed for TNFA, IL18 and ACTB by TaqMan real-time PCR. Fluorescence data were analysed by the regular CT method with a standard curve, and by NLR with a positive control for conversion of fluorescence intensity to copy number, and for this purpose an automated algorithm was written in SPSS syntax. Eleven separate regression mod...Continue Reading

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Citations

May 5, 2007·Journal of Cancer Research and Clinical Oncology·She-Juan AnYi-Long Wu
Oct 21, 2011·Nucleic Acids Research·Tzachi BarAles Tichopad
Apr 14, 2010·BMC Bioinformatics·Davide SistiVilberto Stocchi
Apr 21, 2007·BMC Bioinformatics·Yann KarlenNicolas Mermod
Oct 25, 2007·BMC Bioinformatics·Marjo V SmithChris J Portier
Sep 25, 2008·Analytical Biochemistry·Stefan Cikos, Juraj Koppel
Jul 18, 2006·Journal of Theoretical Biology·Nadia Lalam
Dec 1, 2017·Biochemistry and Cell Biology = Biochimie Et Biologie Cellulaire·Charles GaudreaultJoël Sirois
Oct 31, 2007·Statistics in Medicine·Inna ChervonevaTerry Hyslop
Mar 19, 2009·Scandinavian Journal of Gastroenterology·Trine OlsenJon Florholmen
Sep 14, 2007·Scandinavian Journal of Gastroenterology·Trine OlsenJon Florholmen

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Methods Mentioned

BETA
PCR
biopsies

Software Mentioned

soFAR
MathType
SDS
XP
SPSS

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