Quantitative real-time RT-PCR data analysis: current concepts and the novel "gene expression's CT difference" formula.
For quantification of gene-specific mRNA, quantitative real-time RT-PCR has become one of the most frequently used methods over the last few years. This article focuses on the issue of real-time PCR data analysis and its mathematical background, offering a general concept for efficient, fast and precise data analysis superior to the commonly used comparative CT (DeltaDeltaCT) and the standard curve method, as it considers individual amplification efficiencies for every PCR. This concept is based on a novel formula for the calculation of relative gene expression ratios, termed GED (Gene Expression's CT Difference) formula. Prerequisites for this formula, such as real-time PCR kinetics, the concept of PCR efficiency and its determination, are discussed. Additionally, this article offers some technical considerations and information on statistical analysis of real-time PCR data.
Schefe
Jan H
JH
Center for Cardiovascular Research (CCR)/Institute of Pharmacology and Toxicology, Charité-Universitätsmedizin Berlin, Hessische Strasse 3-4, 10115, Berlin, Germany.
Lehmann
Kerstin E
KE
Buschmann
Ivo R
IR
Unger
Thomas
T
Funke-Kaiser
Heiko
H
D052160:Benzothiazoles; D003627:Data Interpretation, Statistical; D003959:Diamines; D005456:Fluorescent Dyes; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008957:Models, Genetic; D015233:Models, Statistical; D009930:Organic Chemicals; D011804:Quinolines; D012333:RNA, Messenger / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D019914:Taq Polymerase; D013997:Time Factors
D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D012333:RNA, Messenger; C098022:SYBR Green I; D019914:Taq Polymerase