Standardized determination of real-time PCR efficiency from a single reaction set-up.
We propose a computing method for the estimation of real-time PCR amplification efficiency. It is based on a statistic delimitation of the beginning of exponentially behaving observations in real-time PCR kinetics. PCR ground fluorescence phase, non-exponential and plateau phase were excluded from the calculation process by separate mathematical algorithms. We validated the method on experimental data on multiple targets obtained on the LightCycler platform. The developed method yields results of higher accuracy than the currently used method of serial dilutions for amplification efficiency estimation. The single reaction set-up estimation is sensitive to differences in starting concentrations of the target sequence in samples. Furthermore, it resists the subjective influence of researchers, and the estimation can therefore be fully instrumentalized.
Tichopad
Ales
A
Institute of Physiology, FML-Weihenstephan, Center of Life and Food Science, Technical University of Munich, Germany.
Dilger
Michael
M
Schwarz
Gerhard
G
Pfaffl
Michael W
MW
D000465:Algorithms; D000818:Animals; D052160:Benzothiazoles; D002417:Cattle; D004247:DNA / Q000737:chemistry / Q000235:genetics; D003959:Diamines; D005453:Fluorescence; D024282:Genes, sry / Q000235:genetics; D009930:Organic Chemicals / Q000737:chemistry; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods* / Q000592:standards*; D011804:Quinolines; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D004247:DNA