Standardized determination of real-time PCR efficiency from a single reaction set-up

Abstract

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.

Comprehensive Algorithm for Quantitative Real-Time Polymerase Chain Reaction

“…To find a suitable mathematical representation of the complete kinetic curve for each PCR reaction, we compared several equations that generate S-shaped curves that are widely used for fitting enzymatic reactions: four-parameter logistic (Tichopad et al, 2003), sigmoid (Liu and Saint, 2002b;Tichopad et al, 2002Tichopad et al, , 2004, Gompertz (Schlereth et al, 1998), and Chapman models (Glover et al, 1997) (see Table 2). Figure 2A is an example for one typical sample where y 0 is the ground fluorescence value and a is the difference between maximum and ground fluorescence values.…”

“…Other methods have approached this problem including use of the amplification plot (Liu and Saint, 2002a), the first derivative maximum of sigmoid model fitting (Liu and Saint, 2002b;Tichopad et al, 2002), the interactive window-of-linearity algorithm (Ramakers et al, 2003), the mid-value point regression (Peirson et al, 2003), the studentized residual statistics followed by four-parameter logistic model fitting algorithm (FPLM) (Tichopad et al, 2003), and the kinetic outlier detection (KOD) method (Bar et al, 2003). Only the last two methods used statistical techniques to estimate the efficiency objectively.…”

Rationale for the recommendations for harmonizing current methodology for detecting BCR-ABL transcripts in patients with chronic myeloid leukaemia

“…This method is a simplified approach for efficiency calculation as the efficiency may change as the starting copy concentration varies and therefore may not be accurate. 26,27 Nevertheless, the method is useful to compare relative amplification efficiencies. The acceptability of the relative efficiencies can be confirmed by demonstrating that when patient cDNA is diluted in a 10-fold series, the transcript values are not significantly different from the expected 1 log reduction at each dilution step.…”

Aberrant Phenotype and Function of Myeloid Dendritic Cells in Systemic Lupus Erythematosus

“…After denaturation at 95°C for 15 min, 55 cycles were performed at 95°C for 15 s, followed by 60°C for 1 min. Comparative cycle threshold method with PCR efficiency correction, also known as Pfaffl's method, was used for quantification, as previously described (48). The expression levels of the target genes were adjusted to the expression levels of the housekeeping genes PBGD and ␤-actin.…”