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Open Access Highly Accessed Methodology article

Statistical analysis of real-time PCR data

Joshua S Yuan12, Ann Reed3, Feng Chen1 and C Neal Stewart1*

Author Affiliations

1 Department of Plant Sciences, University of Tennessee, Knoxville, TN 37996, USA

2 University of Tennessee Institute of Agriculture Genomics Hub, University of Tennessee, Knoxville, TN 37996, USA

3 Statistical Consulting Center, University of Tennessee, Knoxville, TN 37996, USA

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BMC Bioinformatics 2006, 7:85  doi:10.1186/1471-2105-7-85

Published: 22 February 2006

Abstract

Background

Even though real-time PCR has been broadly applied in biomedical sciences, data processing procedures for the analysis of quantitative real-time PCR are still lacking; specifically in the realm of appropriate statistical treatment. Confidence interval and statistical significance considerations are not explicit in many of the current data analysis approaches. Based on the standard curve method and other useful data analysis methods, we present and compare four statistical approaches and models for the analysis of real-time PCR data.

Results

In the first approach, a multiple regression analysis model was developed to derive ΔΔCt from estimation of interaction of gene and treatment effects. In the second approach, an ANCOVA (analysis of covariance) model was proposed, and the ΔΔCt can be derived from analysis of effects of variables. The other two models involve calculation ΔCt followed by a two group t-test and non-parametric analogous Wilcoxon test. SAS programs were developed for all four models and data output for analysis of a sample set are presented. In addition, a data quality control model was developed and implemented using SAS.

Conclusion

Practical statistical solutions with SAS programs were developed for real-time PCR data and a sample dataset was analyzed with the SAS programs. The analysis using the various models and programs yielded similar results. Data quality control and analysis procedures presented here provide statistical elements for the estimation of the relative expression of genes using real-time PCR.