A New Mathematical Model for Relative Quantification in Real-Time RT-PCR

Nucleic Acids Res. 2001 May 1;29(9):e45. doi: 10.1093/nar/29.9.e45.

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.

Publication types

  • Comparative Study
  • Evaluation Study

MeSH terms

  • Animals
  • DNA Primers
  • Gene Expression Regulation
  • Models, Theoretical*
  • RNA, Messenger / analysis
  • Reference Standards
  • Reproducibility of Results
  • Reverse Transcriptase Polymerase Chain Reaction* / standards
  • Sensitivity and Specificity
  • Time Factors
  • Transcription, Genetic

Substances

  • DNA Primers
  • RNA, Messenger