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Quantification of mitochondrial DNA copy number: pre-analytical factors.

Author(s): Andreu AL, Martinez R, Marti R, García-Arumí E

Publication: Mitochondrion, 2009, Vol. 9, Page 242-246

PubMed ID: 19272467 PubMed Review Paper? No

Purpose of Paper

The purpose of this paper was to identify the contribution of blood product and anticoagulant choice, processing delay, and isolation method on mitochondrial DNA (mtDNA) copy number determination.

Conclusion of Paper

The most significant differences were seen between blood products used for isolation. Peripheral blood mononuclear cells (PBMC) had they highest copy number followed by BC without red blood cells (BCRBC) Delaying processing time by 3 h increased mtDNA yield in all blood products examined. The use of EDTA or citrate as anticoagulants had no effect on mitochondrial DNA copy number. No significant effects on mtDNA yield in BC specimens were identified between three DNA isolation methods, although significant effects on the standard deviations of the three extraction methods were observed. The QIAamp mini kit was the most precise with a coefficient of variance (CV) of 5.7%. The authors recommend that mtDNA copy number reference values be determined with regard to the use of WB, PBMC, BC and BCWRBC and that the QIAamp mini kit be used for all mtDNA isolation.

Studies

  1. Study Purpose

    The purpose of this study was to determine the effects of anticoagulant choice, and the use of whole blood (WB) or buffy coat (BC) mtDNA copy number determination by real-time quantitative RT-PCR (qPCR).

    Summary of Findings:

    Significant differences were seen between blood products used for isolation. Whole blood yielded 101.6 copies mtDNA per copy nuclear DNA whereas BC only had 60.1 copies (p<0.001). The effect was seen regardless of the extraction method used for all biospecimens. The use of EDTA or citrate as anticoagulants had no effect on mitochondrial DNA copy number. Based on the results of this study the authors recommend that mtDNA copy number reference values be specifically determined for WB, versus buffy coat.

    Biospecimens
    Preservative Types
    • None (Fresh)
    Diagnoses:
    • Normal
    Platform:
    AnalyteTechnology Platform
    DNA Real-time qPCR
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Biospecimen Acquisition Anticoagulant Citrate
    EDTA
    Biospecimen Aliquots and Components Blood and blood products Buffy coat
    Whole blood
    View more
  2. Study Purpose

    The purpose of this study was to investigate the effect of DNA isolation method on mtDNA copy number determination from BC by real-time qPCR.

    Summary of Findings:

    The BC from one subject was divided into 30 samples from which mtDNA was extracted using the phenol method, the QIAamp mini kit or the QIAamp Midi kit. No significant effects on mtDNA yield were identified between the 3 extraction methods, but significant effects (p<0.001) on the standard deviations of the three extraction methods were observed. The QIAamp mini kit was the most precise with a coefficient of variance (CV) of 5.7% as compared to a CV of 17.8% for the QIAamp midi kit. The phenol method had a CV of 57.9% due to one outlier. When the outlier was excluded the CV dropped to 5.2%. The authors believe the outlier in the phenol method could be due to problems in the DNA precipitation and resuspension steps which are not present in the other methods investigated. The authors recommend the use of the QIAamp mini kit for mtDNA extraction because of the high degree of reproducibility this kit provides.

    Biospecimens
    Preservative Types
    • None (Fresh)
    Diagnoses:
    • Normal
    Platform:
    AnalyteTechnology Platform
    DNA Real-time qPCR
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Analyte Extraction and Purification Analyte isolation method Phenol method
    QIAamp mini kit (Qiagen)
    QIAamp midi kit (Qiagen)
    View more
  3. Study Purpose

    The purpose of this study was to determine the effects of using peripheral blood mononuclear cells (PBMC), buffy coat (BC) or buffy coat without red blood cells (BCWRBC) as well as delaying processing (0h and 3h) on determination of mitochondrial DNA copy number by real-time qPCR.

    Summary of Findings:

    Peripheral blood mononuclear cells (PBMC) had the highest mitochondrial DNA copy number (181-229.9 copies) followed by BC without red blood cells (BCRBC) (129-9-154.4 copies). Buffy coat only produced (68.1-88.7) copies mtDNA per copy nuclear DNA. The authors attribute the increased mtDNA content in the peripheral blood mononuclear cells to platelet enrichment during separation because of the similar densities of red blood cells and platelets. Delaying processing by 3 h increased mtDNA yield in all blood products examined but the yield increase ranged from 8-66% depending on biospecimen. For most biospecimens the effects was an approximately 20% increase in mtDNA yield after a 3 h incubation. Based on these results the use of PBMC and a 3 hour incubation prior to processing is recommended.

    Biospecimens
    Preservative Types
    • None (Fresh)
    Diagnoses:
    • Normal
    Platform:
    AnalyteTechnology Platform
    DNA Real-time qPCR
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Biospecimen Aliquots and Components Blood and blood products Buffy coat
    Buffy coat without red blood cells
    Peripheral blood mononuclear cells
    Storage Time at room temperature 0 h
    3 h
    View more

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