Using ΦX174 DNA as an exogenous reference for measuring mitochondrial DNA copy number

Quantitative real-time PCR has become a popular method to analyze and quantify changes in the copy number of mitochondrial DNA (mtDNA), and nuclear DNA (nDNA) is often used as an endogenous reference for mtDNA abundance. In our experience, using nDNA as a reference is problematic, due to differences...

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Published inBioTechniques Vol. 47; no. 4; pp. 867 - 869
Main Authors Myers, Meagan B, Mittelstaedt, Roberta A, Heflich, Robert H
Format Journal Article
LanguageEnglish
Published Natick, MA Future Science Ltd 01.10.2009
Eaton
Taylor & Francis Group
Subjects
Biological and medical sciences
Diverse techniques
DNA extraction
Fundamental and applied biological sciences. Psychology
mitochondrial DNA copy number
Molecular and cellular biology
quantitative PCR
ΦX174 DNA
Exogenous
Mitochondrial DNA
Copy number
Reference
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Abstract Quantitative real-time PCR has become a popular method to analyze and quantify changes in the copy number of mitochondrial DNA (mtDNA), and nuclear DNA (nDNA) is often used as an endogenous reference for mtDNA abundance. In our experience, using nDNA as a reference is problematic, due to differences in the extraction efficiency of nDNA and mtDNA and variation in the ploidy of experimental samples. Here, we report that the ratio of mtDNA to nDNA varies in repeated DNA extractions but that ΦX174 DNA, added before DNA extraction, is extracted with a similar efficiency to mtDNA, making it a suitable alternative reference for quantifying mtDNA copy number.
AbstractList Quantitative real-time PCR has become a popular method to analyze and quantify changes in the copy number of mitochondrial DNA (mtDNA), and nuclear DNA (nDNA) is often used as an endogenous reference for mtDNA abundance. In our experience, using nDNA as a reference is problematic, due to differences in the extraction efficiency of nDNA and mtDNA and variation in the ploidy of experimental samples. Here, we report that the ratio of mtDNA to nDNA varies in repeated DNA extractions but that ΦX174 DNA, added before DNA extraction, is extracted with a similar efficiency to mtDNA, making it a suitable alternative reference for quantifying mtDNA copy number.
Author Mittelstaedt, Roberta A
Myers, Meagan B
Heflich, Robert H
AuthorAffiliation 1Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, US Food and Drug Administration, Jefferson, AR, USA
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SubjectTerms Biological and medical sciences
Diverse techniques
DNA extraction
Fundamental and applied biological sciences. Psychology
mitochondrial DNA copy number
Molecular and cellular biology
quantitative PCR
ΦX174 DNA
Title Using ΦX174 DNA as an exogenous reference for measuring mitochondrial DNA copy number
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