Mitochondrial DNA as a non-invasive biomarker: Accurate quantification using real time quantitative PCR without co-amplification of pseudogenes and dilution bias

Malik, Afshan N.; Shahni, Rojeen; Rodriguez-de-Ledesma, Ana; Laftah, Abas H.; Cunningham, Phil

doi:10.1016/j.bbrc.2011.06.067

Cited by 123 publications

(111 citation statements)

References 52 publications

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“…The template DNA was sonicated, the concentration adjusted to 10ng/ul, and qPCR was carried out in triplicate for 10 ng and 1 ng per template, the resulting averages representing 6 measurements per original sample for each gene were used to calculate the mitochondrial genome to nuclear genome ratio in the samples (Table 2) and to test the accuracy of the assay (Figure 2a). The amount of mitochondrial and nuclear DNA in 10ng and 1ng, using values extrapolated from the standards curves, is shown in figures 2b and 2c respectively and shows that as expected, 10 fold reduction of the amount of template used results in 10fold reduction of both MtDNA and B2M, i.e removal of the dilution effect which can skew the Mt/N ratio [16]. The Mt/N values in the samples remain constant ( Figure 2a) and melting curve analysis and DNA sequencing confirmed the specificity of the qPCR reactions (not shown).…”

Section: Absolute Quantification Of Mouse Mitochondrial Dnasupporting

confidence: 65%

“…Unique regions were identified in the mouse mitochondrial sequence, retrieved from ENSEMBL (19) using FASTA version 3.5.2.7 (20) as described previously (16). To measure nuclear DNA, primers against single copy nuclear gene, Beta-2 microglobulin (B2M) were designed (Table 1).…”

Section: 1identification Of Unique Regions Of Mitochondrial Genomementioning

confidence: 99%

“…Total genomic DNA was extracted using the DNeasy Blood & Tissue kit according to manufacturer"s instruction (Qiagen) (16). Before proceeding to qPCR, the DNA template was subjected to the pre-treatment (DNA template shearing using bath sonicator Kerry, Pulsatron 55 which uses 38kHz+/-for 10 minutes) as described previously (15,16) in order to avoid dilution bias. The template concentration was determined using NanoDrop and adjusted to 10ng/μl.…”

Section: Genomic Dna Preparationmentioning

confidence: 99%

“…The most commonly used method used measure MtDNA content in a cell is to quantify the mitochondrial genome versus nuclear genome ratio, termed Mt/N (7,(14)(15)(16) MtDNA from human samples (16). However, a similar approach has not been described for mouse MtDNA and there is little information in the literature about whether mouse MtDNA is present as NumtS in the nuclear genome.…”

Section: Introductionmentioning

confidence: 99%

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Accurate quantification of mouse mitochondrial DNA without co-amplification of nuclear mitochondrial insertion sequences

2016

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(2016). Accurate quantification of mouse mitochondrial DNA without co-amplification of nuclear mitochondrial insertion sequences. MITOCHONDRION. DOI: 10.1016DOI: 10. /j.mito.2016 Citing this paper Please note that where the full-text provided on King's Research Portal is the Author Accepted Manuscript or Post-Print version this may differ from the final Published version. If citing, it is advised that you check and use the publisher's definitive version for pagination, volume/issue, and date of publication details. And where the final published version is provided on the Research Portal, if citing you are again advised to check the publisher's website for any subsequent corrections. General rightsCopyright and moral rights for the publications made accessible in the Research Portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognize and abide by the legal requirements associated with these rights.•Users may download and print one copy of any publication from the Research Portal for the purpose of private study or research.•You may not further distribute the material or use it for any profit-making activity or commercial gain •You may freely distribute the URL identifying the publication in the Research Portal Take down policyIf you believe that this document breaches copyright please contact librarypure@kcl.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT AbbreviationsMtDNA-mitochondrial DNA NumtS-nuclear mitochondrial insertion sequence A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT2 Abstract:Background: Mitochondria contain extra-nuclear genome in the form of mitochondrial DNA (MtDNA), damage to which can lead to inflammation and bioenergetic deficit. Changes inMtDNA levels are increasingly used as a biomarker of mitochondrial dysfunction. We previously reported that in humans, fragments in the nuclear genome known as nuclear mitochondrial insertion sequences (NumtS) affect accurate quantification of MtDNA. In the current paper our aim was to determine whether mouse NumtS affect the quantification ofMtDNA and to establish a method designed to avoid this. Methods:The existence of NumtS in the mouse genome was confirmed using blast N, unique MtDNA regions were identified using FASTA, and MtDNA primers which do not co-amplify NUMTs were designed and tested. MtDNA copy numbers were determined in a range of mouse tissues as the ratio of the mitochondrial and nuclea...

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Section: Absolute Quantification Of Mouse Mitochondrial Dnasupporting

confidence: 65%

Section: 1identification Of Unique Regions Of Mitochondrial Genomementioning

confidence: 99%

Section: Genomic Dna Preparationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Accurate quantification of mouse mitochondrial DNA without co-amplification of nuclear mitochondrial insertion sequences

2016

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“…Briefly, we isolated genomic DNA from buffy coat samples and hybridized the DNA to Affymetrix 6.0 microarrays according to the manufacturer's protocol. Because sequence similarity between mitochondrial and nuclear DNA can adversely affect the accuracy of mtDNA copy number estimates, 39 we curated 25 high-quality mitochondrial SNPs having probes with a perfect match to the annotated mitochondrial location. We used the median of the normalized intensity (log R ratio) for homozygous calls of these 25 mitochondrial SNPs corrected for GC content 40 as the initial estimate of mtDNA copy number.…”

Section: Measures Of Mtdna Copy Numbermentioning

confidence: 99%

Association between Mitochondrial DNA Copy Number in Peripheral Blood and Incident CKD in the Atherosclerosis Risk in Communities Study

Tin

Grams²,

Ashar

et al. 2016

JASN

118

115

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Mitochondrial dysfunction in kidney cells has been implicated in the pathogenesis of CKD. Mitochondrial DNA (mtDNA) copy number is a surrogate measure of mitochondrial function, and higher mtDNA copy number in peripheral blood has been associated with lower risk of two important risk factors for CKD progression, diabetes and microalbuminuria. We evaluated whether mtDNA copy number in peripheral blood associates with incident CKD in a population-based cohort of middle-aged adults. We estimated mtDNA copy number using 25 high-quality mitochondrial single nucleotide polymorphisms from the Affymetrix 6.0 array. Among 9058 participants, those with higher mtDNA copy number had a lower rate of prevalent diabetes and lower C-reactive protein levels and white blood cell counts. Baseline eGFR did not differ significantly by mtDNA copy number. Over a median follow-up of 19.6 years, 1490 participants developed CKD. Higher mtDNA copy number associated with lower risk of incident CKD (highest versus lowest quartile: hazard ratio 0.65; 95% confidence interval, 0.56 to 0.75; P,0.001) after adjusting for age, sex, and race. After adjusting for additional risk factors of CKD, including prevalent diabetes, hypertension, C-reactive protein level, and white blood cell count, this association remained significant (highest versus lowest quartile: hazard ratio 0.75; 95% confidence interval, 0.64 to 0.87; P,0.001). In conclusion, higher mtDNA copy number associated with lower incidence of CKD independent of traditional risk factors and inflammation biomarker levels in this cohort. Further research on modifiable factors influencing mtDNA copy number may lead to improvement in the prevention and treatment of CKD.

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Mitochondrial DNA as a Potential Translational Biomarker of Mitochondrial Dysfunction in Drug‐Induced Toxicity Studies

Malik

2018

Mitochondrial Dysfunction Caused by Drugs and Environmental Toxicants

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Mitochondrial DNA as a non-invasive biomarker: Accurate quantification using real time quantitative PCR without co-amplification of pseudogenes and dilution bias

Cited by 123 publications

References 52 publications

Accurate quantification of mouse mitochondrial DNA without co-amplification of nuclear mitochondrial insertion sequences

Accurate quantification of mouse mitochondrial DNA without co-amplification of nuclear mitochondrial insertion sequences

Association between Mitochondrial DNA Copy Number in Peripheral Blood and Incident CKD in the Atherosclerosis Risk in Communities Study

Mitochondrial DNA as a Potential Translational Biomarker of Mitochondrial Dysfunction in Drug‐Induced Toxicity Studies

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