Accurate Estimation of Nucleic Acids by Amplification Efficiency Dependent PCR
Open Access
- 17 August 2012
- journal article
- research article
- Published by Public Library of Science (PLoS) in PLOS ONE
- Vol. 7 (8), e42063
- https://doi.org/10.1371/journal.pone.0042063
Abstract
Accurate estimation of template - DNA or RNA by real time PCR is dependent on the amplification efficiency (F) of the reaction. The analytical equation describing the kinetics of PCR that is influenced by template re-annealing is formulated. It predicts the gradual reduction of F - from its initial value of 2, leading to template saturation. From an experimental standpoint, due to the exponential nature of the reaction a minute change in F can lead to a large error in the estimation of the initial template concentration. On the basis of individual variation in the amplification efficiency we have formulated a simple mathematical model and an MS Excel based data analysis software that allows accurate and automated quantification of initial template concentration. This method which does not require any normalisation with housekeeping genes was validated by transcript profiling of the genes in the TCA/glyoxylate cycle of E. coli. Consistent with published reports, we observed a precise and specific induction of the glyoxylate shunt genes when the bacteria was shifted from a six carbon glucose media to a two carbon source like acetate.Keywords
This publication has 42 references indexed in Scilit:
- Enhanced analysis of real-time PCR data by using a variable efficiency model: FPK-PCRNucleic Acids Research, 2011
- Real-time PCR quantification using a variable reaction efficiency modelAnalytical Biochemistry, 2008
- Highly accurate sigmoidal fitting of real-time PCR data by introducing a parameter for asymmetryBMC Bioinformatics, 2008
- Statistical methods for efficiency adjusted real‐time PCR quantificationBiotechnology Journal, 2008
- Statistical tools for transgene copy number estimation based on real-time PCRBMC Bioinformatics, 2007
- Comprehensive Algorithm for Quantitative Real-Time Polymerase Chain ReactionJournal of Computational Biology, 2005
- Mathematical model of real‐time PCR kineticsBiotechnology & Bioengineering, 2005
- Standardized determination of real-time PCR efficiency from a single reaction set-upNucleic Acids Research, 2003
- Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT MethodMethods, 2001
- A new mathematical model for relative quantification in real-time RT-PCRNucleic Acids Research, 2001