Accurate Measurement of Mitochondrial DNA Deletion Level and Copy Number Differences in Human Skeletal Muscle

<div><p>Accurate and reliable quantification of the abundance of mitochondrial DNA (mtDNA) molecules, both wild-type and those harbouring pathogenic mutations, is important not only for understanding the progression of mtDNA disease but also for evaluating novel therapeutic approaches. A clear understanding of the sensitivity of mtDNA measurement assays under different experimental conditions is therefore critical, however it is routinely lacking for most published mtDNA quantification assays. Here, we comprehensively assess the variability of two quantitative Taqman real-time PCR assays, a widely-applied <i>MT-ND1</i>/<i>MT-ND4</i> multiplex mtDNA deletion assay and a recently developed <i>MT-ND1</i>/<i>B2M</i> singleplex mtDNA copy number assay, across a range of DNA concentrations and mtDNA deletion/copy number levels. Uniquely, we provide a specific guide detailing necessary numbers of sample and real-time PCR plate replicates for accurately and consistently determining a given difference in mtDNA deletion levels and copy number in homogenate skeletal muscle DNA.</p></div

Inter- and intra-plate variability in the measurement of mtDNA copy number differences between DNA samples.

<p>Replicate (<i>n</i> = 12) <i>MT</i>-<i>ND1</i>/<i>B2M</i> copy number ratios were obtained for five homogenate skeletal muscle DNA samples of varying mtDNA copy number (samples S1 to S5) on each of four replicate <i>MT</i>-<i>ND1</i>/<i>B2M</i> real time PCR plate pairs at different DNA concentrations (low – corresponding to an average C_{q<i>MT-ND1</i>} of 19 and an average C_q<i>B2M</i> of 27, moderate - C_{q<i>MT-ND1</i>} = 24 and C_q<i>B2M</i> = 30, high - C_{q<i>MT-ND1</i>} = 25.5 and C_q<i>B2M</i> = 33). Independent relative copy number values were then calculated for each replicate of S2, S3, S4 and S5 compared to S1 for each <i>MT</i>-<i>ND1</i>/<i>B2M</i> plate. Intra-plate standard deviations are shown as circles (relative copy number values: S2:S1 = 1.10 (green circles), S3:S1 = 1.25 (blue circles), S4:S1 = 1.50 (orange circles), S5:S1 = 1.9 (red circles)). Inter-plate standard deviations are shown as grey crosses. As relative copy number is a multiplicative relationship, geometric standard deviations are shown.</p

Clinical details of patients with single, large-scale mtDNA deletions and control individuals.

<p>CPEO, chronic progressive external ophthalmoplegia.</p>1<p>Age at time of biopsy.</p>2<p>GenBank accession number NC_012920.1.</p>3<p>Patient 1 published in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114462#pone.0114462-Murphy1" target="_blank">[7]</a>.</p>4<p>Patient 3 published in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114462#pone.0114462-Taivassalo1" target="_blank">[8]</a>.</p><p>Clinical details of patients with single, large-scale mtDNA deletions and control individuals.</p

Number of plates required to identify a given heteroplasmy difference between two homogenate skeletal muscle DNA samples at various <i>MT</i>-<i>ND4</i> deletion levels with the <i>MT</i>-<i>ND1</i>/<i>MT</i>-<i>ND4</i> Taqman deletion assay.

<p>Standard deviation (SD) is expressed in percentage points.</p><p>Actual power (P) and number of plates (<i>n</i>) are shown. C_q<23 ([DNA]_PCR>1 pg/µl).</p><p>Number of plates required to identify a given heteroplasmy difference between two homogenate skeletal muscle DNA samples at various <i>MT</i>-<i>ND4</i> deletion levels with the <i>MT</i>-<i>ND1</i>/<i>MT</i>-<i>ND4</i> Taqman deletion assay.</p

Effects of DNA concentration and heteroplasmy level on the accuracy of the <i>MT</i>-<i>ND1</i>/<i>MT</i>-<i>ND4</i> deletion assay.

<p>Percentage standard deviations of DNA sample replicates (<i>n</i> = 84) are represented relative to average C_q for homogenate skeletal muscle DNA samples (<i>n</i> = 6) varying in <i>MT</i>-<i>ND4</i> deletion levels from 10–80%. Confidence intervals at 95% are shown. The y axis is log₁₀ scaled.</p

Number of <i>MT</i>-<i>ND1</i> and <i>B2M</i> real time PCR plates required to detect a specific relative difference in mtDNA copy number between two homogenate skeletal muscle DNA samples for a given number of replicate sample wells.

<p>Each cell defines the number of plates (<i>n</i>) and actual power (P; minimum power 0.8 at α = 0.05) for a two sample <i>t</i>-test to detect, with the specified number of sample replicates per plate, a given percentage difference in mtDNA copy number. Estimates are calculated at high (approximate C_{q<i>MT-ND1</i>} of 19 ([DNA]_PCR = 20 pg/µl) and C_q<i>B2M</i> of 27 ([DNA]_PCR = 0.6 ng/µl)), moderate (approximate C_{q<i>MT-ND1</i>} of 24 ([DNA]_PCR = 0.6 pg/µl) and C_q<i>B2M</i> of 30 ([DNA]_PCR = 0.1 ng/µl)) and low (approximate C_{q<i>MT-ND1</i>} of 25 ([DNA]_PCR = 0.3 pg/µl) and C_q<i>B2M</i> of 33 ([DNA]_PCR = 10 pg/µl)) PCR DNA concentration levels.</p><p>Number of <i>MT</i>-<i>ND1</i> and <i>B2M</i> real time PCR plates required to detect a specific relative difference in mtDNA copy number between two homogenate skeletal muscle DNA samples for a given number of replicate sample wells.</p

Inter- and intra-plate variability in the measurement of <i>MT</i>-<i>ND4</i> deletion level differences between DNA samples.

<p>Independent values for the difference in deletion level (in percentage points) between two homogenate skeletal muscle DNA samples harbouring either low (20–25%; samples A–C) or high (70–80%; samples D–F) <i>MT</i>-<i>ND4</i> deletion levels are shown for each sample replicate (<i>n</i> = 24; grey dots) per replicate real time PCR plate (<i>n</i> = 3). Data are normalised by subtracting the best estimate of the true deletion level difference, determined as the mean of all replicates on all plates. Mean values for each dataset are displayed (horizontal bars). The mean values from individual plates are within 1% of the actual deletion level, though individual replicates vary by up to ±9% at low deletion level and ±6% at high deletion level.</p