Real‐Time Quantitative PCR Analysis of Mitochondrial DNA Content

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.