A new mathematical model for relative quantification in real-time RT–PCR

MW Pfaffl - Nucleic acids research, 2001 - academic.oup.com
Nucleic acids research, 2001academic.oup.com
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 …
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.
Oxford University Press