An improvement of the 2ˆ(-delta delta CT) method for quantitative real-time polymerase chain reaction data analysis.

Abstract

Background: The 2^-ΔΔCT method has been extensively used as a relative quantification strategy for quantitative real-time polymerase chain reaction (qPCR) data analysis. This method is a convenient way to calculate relative gene expression levels between different samples in that it directly uses the threshold cycles (CTs) generated by the qPCR system for calculation. However, this approach relies heavily on an invalid assumption of 100% PCR amplification efficiency across all samples. In addition, the 2^-ΔΔCT method is applied to data with automatic removal of background fluorescence by the qPCR software. Since the background fluorescence is unknown, subtracting an inaccurate background can lead to distortion of the results. To address these problems, we present an improved method, the individual efficiency corrected calculation.

Results: Our method takes into account the PCR efficiency of each individual sample. In addition, it eliminates the need for background fluorescence estimation or subtraction because the background can be cancelled out using the differencing strategy. The DNA amount for a certain gene and the relative DNA amount among different samples estimated using our method were closer to the true values compared to the results of the 2^-ΔΔCT method.

Conclusions: The improved method, the individual efficiency corrected calculation, produces more accurate estimates in relative gene expression than the 2^-ΔΔCT method and is thus a better way to calculate relative gene expression.