From the \boldsymbol2^-\boldsymbol\Delta\boldsymbol\Delta \boldsymbolC_\bfT Method to the \boldsymbol2^-\boldsymbolC_\bfT Method: A More Rigorous Approach to Real-time Quantitative Polymerase Chain Reaction Data Analysis

Objective To optimize the real-time quantitative polymerase chain reaction (RT-qPCR) data analysis process through mathematical principles by replacing the biased 2^-\Delta\Delta C_\mathrmT method with a more rigorous 2^-C_\mathrmT method, thereby improving the accuracy of gene expression quantification analysis.

Methods Essentially, the C_T value serves as the exponent in a base-2 exponential equation within the logic of comparative C_T method. In the traditional 2^-\Delta\Delta C_\mathrmT method, the arithmetic means of raw C_T and ΔC_T values are directly calculated and the exponential nature of C_T data is overlooked, which may introduce systematic bias to the calculation results. We propose a new method, entitled the 2^-C_\mathrmT method, in which all calculations are based on the transformation of C_T values into 2^-C_\mathrmT . This includes computing the relative initial expression levels of target and reference genes within each sample, the relative abundance of the target gene, and its fold change across groups. Statistical comparisons are then performed based on fold change values. By strictly adhering to the exponential nature of of C_T values, the biases introduced by arithmetic averaging at the C_T or ΔC_T level are avoided. We applied this method to multiple RT-qPCR datasets to evaluate the differences between the traditional 2^-\Delta\Delta C_\mathrmT and the proposed 2^-C_\mathrmT methods in gene expression quantification, as well as the effect of the differences.

Results In the original dataset from LIVAK and SCHMITTGEN, the two methods produced similar results. However, in the cadmium exposure experiment, findings from the 2^-\Delta\Delta C\mathrm_T method indicated that 8-hour cadmium exposure caused an increase of irg-6 gene expression in Caenorhabditis elegans from 1.314-fold to 7.125-fold (P = 0.0002). In contrast, findings from the 2^-C_\mathrmT method showed a fold change from 1.0 to 4.124 (P = 0.0015), a 70% difference between the two methods.

Conclusion The 2^-C_\mathrmT method provides a mathematically more rigorous approach that more accurately reflects gene expression changes, particularly in experiments with high C_T variability. It offers a more reliable computational paradigm for quantitative gene expression analysis.