Quantifying mitochondrial DNA copy number using robust regression to interpret real time PCR results

Abstract

Background: Real time PCR (rtPCR) is a quantitative assay to determine the relative DNA copy number in a sample versus a reference. The $$\Delta C-T$$ Δ C T method is the standard for the analysis of the output data generated by an rtPCR experiment. We developed an alternative based on fitting a robust regression to the rtPCR signal. This new data analysis tool reduces potential biases and does not require all of the compared DNA fragments to have the same PCR efficiency. Results: Comparing the two methods when analysing 96 identical PCR preparations showed similar distributions of the estimated copy numbers. Estimating the efficiency with the $$\Delta C-T$$ Δ C T method, however, required a dilution series, which is not necessary for the robust regression method. We used rtPCR to quantify mitochondrial DNA (mtDNA) copy numbers in three different tissues types: breast, colon and prostate. For each type, normal tissue and a tumor from the same three patients were analysed. This gives a total of six samples. The mitochondrial copy number is estimated to lie between 200 and 300 copies per cell. Similar results are obtained when using the robust regression or the $$\Delta C-T$$ Δ C T method. Confidence ratios were slightly narrower for the robust regression. The new data analysis method has been implemented as an R package.