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A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res
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@MISC{Pfaffl_anew,
author = {Michaelw. Pfaffl},
title = {A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res},
year = {}
}
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Abstract
Use of the real-time polymerase chain reaction (PCR) to amplify cDNA products reverse transcribed from mRNA is on the way to becoming a routine tool in molecular biology to study low abundance gene expression. Real-time PCR is easy to perform, provides the necessary accuracy and produces reliable as well as rapid quantification results. But accurate quantification of nucleic acids requires a reproducible methodology and an adequate mathematical model for data analysis. This study enters into the particular topics of the relative quantification in real-time RT–PCR of a target gene transcript in comparison to a reference gene transcript. Therefore, a new mathematical model is presented. The relative expression ratio is calculated only from the real-time PCR efficiencies and the crossing point deviation of an unknown sample versus a control. This model needs no calibration curve. Control levels were included in the model to standardise each reaction run with respect to RNA integrity, sample loading and inter-PCR variations. High accuracy and reproducibility (<2.5 % variation) were reached in LightCycler PCR using the established mathematical model.
Keyphrases
new mathematical model relative quantification real-time rt-pcr nucleic acid re target gene transcript crossing point deviation control level accurate quantification sample loading high accuracy inter-pcr variation cdna product real-time rt pcr real-time pcr efficiency reproducible methodology data analysis molecular biology unknown sample lightcycler pcr mathematical model routine tool real-time pcr relative expression ratio real-time polymerase chain reaction particular topic reference gene transcript necessary accuracy low abundance gene expression rna integrity rapid quantification result adequate mathematical model calibration curve nucleic acid
