A common base method for analysis of qPCR data and the application of simple blocking in qPCR experiments

Abstract

Background: qPCR has established itself as the technique of choice for the quantification of gene expression. Procedures for conducting qPCR have received significant attention; however, more rigorous approaches to the statistical analysis of qPCR data are needed.

Results: Here we develop a mathematical model, termed the Common Base Method, for analysis of qPCR data based on threshold cycle values (C _q ) and efficiencies of reactions (E). The Common Base Method keeps all calculations in the logscale as long as possible by working with log₁₀(E) ∙ C _q , which we call the efficiency-weighted C _q value; subsequent statistical analyses are then applied in the logscale. We show how efficiency-weighted C _q values may be analyzed using a simple paired or unpaired experimental design and develop blocking methods to help reduce unexplained variation.

Conclusions: The Common Base Method has several advantages. It allows for the incorporation of well-specific efficiencies and multiple reference genes. The method does not necessitate the pairing of samples that must be performed using traditional analysis methods in order to calculate relative expression ratios. Our method is also simple enough to be implemented in any spreadsheet or statistical software without additional scripts or proprietary components.

Keywords: Analysis of variance (ANOVA); Blocking; Confidence intervals; Paired and unpaired tests; Statistics; qPCR analysis.

Fig. 1

Origin of the Efficiency (E) and C _q values. Δ$C_q^{\left(w\right)}$ values are derived from the arithmetic means of the technical replicates. Inset A shows the derivation of sample types A and B in an unpaired sample test where sample types derive from different biological replicates. Inset B shows the derivation of sample types A and B in a paired sample test where sample types derive from the same biological replicate. Please note that each E value is logtransformed and multiplied by C _q as discussed in the text. This transformation is not shown in the interest of saving space

Fig. 2

Presentation of results as mean with 95% confidence interval. The results of an unpaired t-test using data from Table 2 are graphically shown. The relative expression ratio of the GOI is plotted along with the 95% confidence interval