From the 2-??CT Method to the 2-CT Method:A More Rigorous Approach to Real-time Quantitative Polymerase Chain Reaction Data Analysis
- VernacularTitle:从2-??CT法到2-CT法:一种更严谨的RT-qPCR数据分析策略
- Author:
Lixiang FENG
1
;
Rongqian ZHAO
;
Kui ZHANG
;
Wenxing YANG
Author Information
- Keywords: RT-qPCR; Data analysis; 2-??CT; 2-CT; Gene expression quantification; Caenorhabditis elegans; Cadmium; irg-6
- From: Journal of Sichuan University (Medical Sciences) 2025;56(5):1405-1411
- CountryChina
- Language:Chinese
- Abstract: Objective To optimize the real-time quantitative polymerase chain reaction(RT-qPCR)data analysis process through mathematical principles by replacing the biased 2-??CT method with a more rigorous 2-CT method,thereby improving the accuracy of gene expression quantification analysis.Methods Essentially,the CT value serves as the exponent in a base-2 exponential equation within the logic of comparative CT method.In the traditional 2-??CT method,the arithmetic means of raw CT and ΔCT values are directly calculated and the exponential nature of CT data is overlooked,which may introduce systematic bias to the calculation results.We propose a new method,entitled the 2-CT method,in which all calculations are based on the transformation of CT values into 2-CT.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 CT values,the biases introduced by arithmetic averaging at the CT or ΔCT level are avoided.We applied this method to multiple RT-qPCR datasets to evaluate the differences between the traditional 2-??CT and the proposed 2-CT 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-??CT 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=0.000 2).In contrast,findings from the 2-CT method showed a fold change from 1.0 to 4.124(P=0.001 5),a 70%difference between the two methods.Conclusion The2-CT method provides a mathematically more rigorous approach that more accurately reflects gene expression changes,particularly in experiments with high CT variability.It offers a more reliable computational paradigm for quantitative gene expression analysis.
