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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PDF or HTML at Wiley Online LibraryTable of Contents
- 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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PDF or HTML at Wiley Online LibraryMaterials
Basic Protocol 1: Real‐Time Quantitative PCR for the Quantification of mtDNA Content
Materials
Support Protocol 1: Generation of mtDNA Age‐Matched Controls
Materials
|
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online LibraryFigures
-
Figure 19.7.1 Cycle parameters utilized for amplification by qPCR. Annealing temperatures can vary according to different T m . View Image -
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 . View Image -
Figure 19.7.3 Control mtDNA content across blood and muscle samples by age. Reprinted with permission from Clinical Chemistry (Dimmock et al., ). View Image
Videos
Literature Cited
| Literature Cited | |
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