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

BMC Research Notes, Nov 2017

Background Real time PCR (rtPCR) is a quantitative assay to determine the relative DNA copy number in a sample versus a reference. The \(\Delta C_T\) 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 \(\Delta C_T\) 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 \(\Delta C_T\) method. Confidence ratios were slightly narrower for the robust regression. The new data analysis method has been implemented as an R package.

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

Refinetti et al. BMC Res Notes Quantifying mitochondrial DNA copy number using robust regression to interpret real time PCR results Paulo Refinetti 0 3 David Warren 2 Stephan Morgenthaler 0 3 Per O. Ekstrøm 1 0 Ecole Polytechnique Féderale de Lausanne , 1015 Lausanne , Switzerland 1 Department of Tumor Biology , Radiumhospital, 0379 Oslo , Norway 2 Department of Medical Biochemistry , Radiumhospital, 0379 Oslo , Norway 3 Ecole Polytechnique Féderale de Lausanne , 1015 Lausanne , Switzerland Background: Real time PCR (rtPCR) is a quantitative assay to determine the relative DNA copy number in a sample versus a reference. The CT 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 CT 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 CT method. Confidence ratios were slightly narrower for the robust regression. The new data analysis method has been implemented as an R package. rtPCR; Robust regression; Mitochondrial DNA Background Mitochondria are the organelle responsible for most of the energy production in eukaryotic cells. Each mitochondrion carries several copies of mitochondrial DNA, which is composed of a single circular chromosome of 16569 base pairs (hg38, GRCh38, Dec. 2013). It encodes for 22 tRNA, 13 protein subunits and two ribosomal RNA subunits. There are currently few accurate measurements of mtDNA copy number in cells [ 1–5 ], even though this number affects the symptoms of mitochondrial diseases [ 6–9 ]. Better measurements of mtDNA copy numbers would improve the understanding of mtDNA mutagenesis [ 10–12 ] as well as the process through which mutations become homoplasmic. Mitochondrial mutations also appear to be involved in cancer development [ 13–17 ], and aging [ 18–21 ]. Furthermore, most tumors are thought to rely on glycolysis rather than oxidative phosphorylation for the majority of their energy, a process that could be related to mtDNA copy number. The standard method for quantifying DNA copy number is real time PCR (rtPCR) [ 22– 24 ]. Most methods rely on amplifying a mitochondrial and a nuclear fragment in separate reactions, with the template from the same sample [ 16, 24 ]. Although there has been much development in the data analysis algorithms applied to rtPCR output, some challenges remainx [ 25, 26 ]. Materials and methods Tissue and DNA extraction Anonymous surgical discards were obtained after standardised informed consent. Tissue was stored at the surgical department at − 70 °C until DNA extraction. Normal and tumor tissue was obtained from three different patients with three different tumor types (breast, prostate and colon). The normal tissue was taken at a distance of 10–15 cm from the location of the tumor. A few milligrams were taken from each sample and had their DNA extracted. DNA extraction Samples were digested with proteinase K for 4 h at 57 °C in 300 µ l of digestion Buffer (Qiagen, Hilden, Germany) according to manufacturer’s instructions. DNA was extracted from them using the Qiagen MagAttract DNA Mini-M48 Kit with a dedicated automatic solution also provided by Qiagen. The result is a DNA solution containing approximately 50 ng of DNA per µ l . Primers Primers were designed using the rtPCR primer design tool of IDT (integrated DNA technologies). The nuclear and mitochondrial primer pairs were designed for simultaneous amplification. Table  1 shows the primer pairs. PCR conditions were optimised by testing various annealing temperatures, reaction volumes, and reagent concentrations. The objective was to use the same conditions for both primers pairs. The mitochondrial primer was chosen so that it could not amplify in the nuclear genome and vice versa. rtPCR condition Real time PCR was performed using a BioRad CFX connect Real-time PCR detection System. The PCR recipe was 2× Perfecta SYBR Green SuperMix for iQ (QuantaBio, Beverly, MA, USA, WHR: 733-1249), 0.2 µ M of each primer, for a final volume of 20 µ l . The PCR temperature cycling used: initial denaturing at 94 °C for 4 min, followed by 45 cycles of denaturing at 94 °C for 30 s, annealing at 60 ° (...truncated)


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Paulo Refinetti, David Warren, Stephan Morgenthaler, Per O. Ekstrøm. Quantifying mitochondrial DNA copy number using robust regression to interpret real time PCR results, BMC Research Notes, 2017, pp. 593, Volume 10, Issue 1, DOI: 10.1186/s13104-017-2913-1