Analyzing real-time PCR data by the comparative C(T) method

Nat Protoc. 2008;3(6):1101-8. doi: 10.1038/nprot.2008.73.


Two different methods of presenting quantitative gene expression exist: absolute and relative quantification. Absolute quantification calculates the copy number of the gene usually by relating the PCR signal to a standard curve. Relative gene expression presents the data of the gene of interest relative to some calibrator or internal control gene. A widely used method to present relative gene expression is the comparative C(T) method also referred to as the 2 (-DeltaDeltaC(T)) method. This protocol provides an overview of the comparative C(T) method for quantitative gene expression studies. Also presented here are various examples to present quantitative gene expression data using this method.

Publication types

  • Comparative Study

MeSH terms

  • Animals
  • Breast Neoplasms / genetics
  • Computer Systems / statistics & numerical data
  • Data Interpretation, Statistical
  • Female
  • Gene Expression
  • Gene Expression Profiling / statistics & numerical data
  • Genes, bcl-2
  • Homeodomain Proteins / genetics
  • Humans
  • Mice
  • MicroRNAs / genetics
  • Pancreas / metabolism
  • Pancreatic Neoplasms / genetics
  • Polymerase Chain Reaction / methods
  • Polymerase Chain Reaction / statistics & numerical data*
  • RNA / genetics
  • RNA, Messenger / genetics
  • Transcription Factors / genetics


  • Homeodomain Proteins
  • MicroRNAs
  • RNA, Messenger
  • Transcription Factors
  • HOXD10 protein, human
  • RNA