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)