Real‐Time Quantitative PCR Analysis of Mitochondrial DNA Content

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

Mitochondrial disorders are a group of complex and heterogeneous diseases that may be caused by molecular defects in the nuclear or mitochondrial genome. The biosynthesis and integrity of the small 16.6?kb mitochondrial genome require a group of nuclear encoded genes. The mitochondrial DNA (mtDNA) depletion syndromes (MDDSs) are autosomal recessive disorders caused by molecular defects in nuclear genes, and characterized by a reduction in mtDNA content. To date, mutations in at least nine genes (POLG , DGUOK , TK2 , TYMP , MPV17 , SUCLA2 , SUCLG1 , RRM2B , and C10orf2 ) have been reported to cause various forms of MDDSs. In the clinical setting, a simple method to determine mtDNA depletion would be useful prior to undertaking gene sequence analysis. This unit outlines the real?time quantitative polymerase chain reaction (qPCR) analysis of mtDNA content in tissues. MtDNA content varies among different tissues and at different ages in the same individual. Detailed protocols for the selection of nuclear genes for normalization, PCR set up, validation procedures, tissue and age matched controls, and sensitivity and specificity in various tissues, as well as interpretation of results are discussed. Curr. Protoc. Hum. Genet. 68:19.7.1?19.7.12 © 2011 by John Wiley & Sons, Inc.

Keywords: mtDNA copy number; mtDNA content; mtDNA qPCR; quantification of mtDNA content; mtDNA depletion

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Introduction
Strategic Planning
Basic Protocol 1: Real‐Time Quantitative PCR for the Quantification of mtDNA Content
Support Protocol 1: Generation of mtDNA Age‐Matched Controls
Support Protocol 2: Preparation and Quantification of DNA Samples
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Real‐Time Quantitative PCR for the Quantification of mtDNA Content

Materials

Genomic DNA samples (see protocol 4 )
qPCR primers for mitochondrial tRNA^Leu(UUR) gene, alternate primers for mtDNA 16S rRNA gene and nuclear β‐2‐microglobulin (β2M ) gene (Table 19.7.2 ), 5 µM working concentration diluted in water
iTaq SYBR Green SuperMix with ROX (Bio‐Rad, cat. no. 170‐8852)
Tissue‐ and age‐matched pooled controls (see protocol 3 )

PCR hood
96‐well thin‐walled PCR plates
Adhesive plate sealer
ABI Prism 7900HT sequence detector system (Applied Biosystems)

Data analysis software: SDS software (version 2.2)

Table 9.7.2 MaterialsqPCR Primer Sequences and Conditions

Gene	Primer name	Primer sequence (5′ to 3′)	Amplicon size (bp)	Annealing temperature (°C)
mtDNA tRNA^Leu(UUR)	tRNA F3212	CACCCAAGAACAGGGTTTGT	107	62
	tRNA R3319	TGGCCATGGGTATGTTGTTA
nDNA β2‐microglobulin	β2M F594	TGCTGTCTCCATGTTTGATGTATCT	86	62
	β2M R679	TCTCTGCTCCCCACCTCTAAGT
Alternate primers:mtDNA 16S rRNA	mtF3163	GCCTTCCCCCGTAAATGATA	97	62
	mtR3260	TTATGCGATTACCGGGCTCT

Support Protocol 1: Generation of mtDNA Age‐Matched Controls

Materials

Tissues
Puregene kit (Gentra Systems, cat. no. D50K1) including:
- Red cell lysis solution
- Cell lysis solution
- DNA hydration solution
- Protein precipitation solution
100% isopropanol
70% ethanol
Rapid hair digestion buffer: 10 mM Tris⋅Cl (pH 8.0), 1.0% Brij 58, 35 mM DTT, 1 mM CaCl₂
Calf thymus DNA standards (type XV; Sigma, cat. no. D4522)
NanoDrop ND‐1000 UV‐Vis spectrophotometer

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Figures

Figure 19.7.1 Cycle parameters utilized for amplification by qPCR. Annealing temperatures can vary according to different T _m .

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Figure 19.7.2 mtDNA copy number amplification plot. The amplification plot represents two patient samples with an age‐matched muscle control. Curves 1 through 3 represent mtDNA amplification of samples with increased mtDNA copy number, normal copy number in an age matched control, and mtDNA depletion, respectively. Curves 4 through 6 represent the amplification of the nDNA using β2M primer set for the same three samples. The results are calculated in Table .

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Figure 19.7.3 Control mtDNA content across blood and muscle samples by age. Reprinted with permission from Clinical Chemistry (Dimmock et al., ).

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Literature Cited

Literature Cited
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