Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR

Xia, Peng; Radpour, Ramin; Zachariah, Rebecca; Fan, Alex Xiu Cheng; Kohler, Corina; Hahn, Sinuhe; Holzgreve, Wolfgang; Zhong, Xiao Yan

doi:10.1590/s1415-47572009000100003

Cited by 32 publications

(30 citation statements)

References 25 publications

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“…Primers and amplification conditions have been described previously (Xia et al, 2009a;Xia et al, 2009b). To determine the quantities of mtDNA and nDNA present in the tested samples, the average threshold cycle (Ct) values for nDNA and mtDNA were obtained from each reaction.…”

Section: Dna Isolation and Multiplex Quantitative Real-time Polymerasmentioning

confidence: 99%

Mitochondrial DNA Levels in Blood and Tissue Samples from Breast Cancer Patients of Different Stages

Xia

Wang²,

Geng³

et al. 2014

Asian Pacific Journal of Cancer Prevention

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Aims: Alterations in mitochondrial DNA (mtDNA) have been implicated in carcinogenesis and tumor progression. We here evaluated the diagnostic and prognostic potential of mtDNA as a biomarker for breast cancer. Methods: Using multiplex real-time polymerase chain reaction, nuclear DNA (nDNA) and mtDNA levels in serum, buffy coat, tumor, and tumor-adjacent tissue samples from 50 breast cancer patients were determined and assessed for associations with clinicopathological features. To evaluate mtDNA as a biomarker for distinguishing between the four sample types, we created receiver operating characteristic (ROC) curves. Results: The mtDNA levels in buffy coat were significantly lower than in other sample types. Relative to tumor-adjacent tissue, reduced levels of mtDNA were identified in buffy coat and tumor tissue but not in serum. According to ROC curve analysis, mtDNA levels could be used to distinguish between buffy coat and tumor-adjacent tissue samples with good sensitivity (77%) and specificity (83%). Moreover, mtDNA levels in serum and tumor tissue were positively associated with cancer TMN stage. Conclusions: The mtDNA levels in blood samples may represent a promising, non-invasive biomarker in breast cancer patients. Additional, large-scale validation studies are required to establish the potential use of mtDNA levels in the early diagnosis and monitoring of breast cancer.

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Section: Dna Isolation and Multiplex Quantitative Real-time Polymerasmentioning

confidence: 99%

Mitochondrial DNA Levels in Blood and Tissue Samples from Breast Cancer Patients of Different Stages

Xia

Wang²,

Geng³

et al. 2014

Asian Pacific Journal of Cancer Prevention

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“…The change in the relative mtDNA copy number was determined as the ratio between the copy number of the mitochondrial tRNA gene and that of the nuclear housekeeping gene GAPDH which was amplified in the same tube (Xia et al 2009). The efficiency of the assay for amplifying both nDNA and mtDNA was measured with standard curves generated by dilution series of 20, 10, 5, 2, 1, and 0.1 ng of total rat liver DNA per reaction.…”

Section: Quantitative Analysis Of Nuclear and Mitochondrial Dna By Rementioning

confidence: 99%

Cell-free DNA in the urine of rats exposed to ionizing radiation

Abdullaev

Minkabirova

Bezlepkin

et al. 2015

Radiat Environ Biophys

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Investigation of cell-free DNA (cf-DNA) in body fluids, as a potential biomarker for assessing the effect of ionizing radiation on the organism, is of considerable interest. We investigated changes in the contents of cell-free mitochondrial DNA (cf-mtDNA) and cell-free nuclear DNA (cf-nDNA) in the urine of X-ray-exposed rats. Assays of cf-mtDNA and cf-nDNA were performed by a real-time PCR in rat urine collected before and after irradiation of animals with doses of 3 and 5 Gy. We also determined the presence of mutations in urine cf-mtDNA, as recognized by Surveyor nuclease. A sharp increase in cf-mtDNA and cf-nDNA in the urine of irradiated rats was observed within 24 h after exposure, followed by a decrease to normal levels. In all cases, the contents of cf-mtDNA fragment copies (estimated by gene tRNA) were significantly higher than those of cf-nDNA estimated by gene GAPDH. A certain portion of mutant cf-mtDNA fragments was detected in the urine of exposed rats, whereas they were absent in the urine of the same animals before irradiation. These preliminary data also suggest that the increased levels of urine cf-mtDNA and cf-nDNA may be a potential biomarker for noninvasive assessment of how the organism responds to ionizing radiation influence.

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“…Therefore, the GE of nDNA and mtDNA were assessed with the multiplex real-time PCR. This process could perform two assays in a single tube at the same time and be an effi cient and accurate method for quantifying the ccf nDNA and mtDNA (14) .…”

Section: Amplifi Cation and Quantifi Cation Of Ndna And Mtdna In Plasmentioning

confidence: 99%

“…The multiplex real-time PCR was performed in a total reaction volume of 25 μ L, containing 5 μ L of DNA, 12.5 μ L of TaqMan Universal PCR Master Mix, four primers and two probes. Our standard TaqMan PCR conditions have been described in our previous publication (14) , which involved a 2 min incubation at 50 ° C, followed by an initial denaturation step at 95 ° C for 10 min and 40 cycles of 1 min at 60 ° C and 15 s at 95 ° C.…”

Section: Amplifi Cation and Quantifi Cation Of Ndna And Mtdna In Plasmentioning

confidence: 99%

“…The three methods were used to select an optimal ccf DNA extraction for reducing input sample volume while increasing the extracted DNA concentration necessary for ccf DNA analysis. The concentration of ccf DNA was measured by Nanodrop and the GE of the GAPDH housekeeping gene and MTATP 8 gene were measured using a multiplex real-time quantitative PCR for the quantifi cation of nDNA and mtDNA, which was successfully developed in our laboratory (14) .…”

Section: Introductionmentioning

confidence: 99%

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Strategies of reducing input sample volume for extracting circulating cell-free nuclear DNA and mitochondrial DNA in plasma

Chen

Cai²,

Zhang³

et al. 2012

Clinical Chemistry and Laboratory Medicine (CCLM)

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Background: Circulating cell-free (ccf) DNA in blood has been suggested as a potential biomarker in many conditions regarding early diagnosis and prognosis. However, misdiagnosis can result due to the limited DNA resources in Biobank ' s plasma samples or insuffi cient DNA targets from a predominant DNA background in genetic tests. This study explored several strategies for an effi cient DNA extraction to increase DNA amount from limited plasma input. Methods: Ccf plasma DNA was extracted with three different methods, a phenol-chloroform-isoamylalcohol (PCI) method, a High Pure PCR Template Preparation Kit method and a method used for single cell PCR in this group. Subsequently, the total DNA was measured by Nanodrop and the genome equivalents (GE) of the GAPDH housekeeping gene and MTATP 8 gene were measured using a multiplex real-time quantitative PCR for the quantitative assessment of nDNA and mtDNA. Results: Instead of 400 -800 μ L (routine input in the laboratory), 50 μ Lof plasma input enabled the extraction of ccf DNA suffi cient for quantitative analysis. Using the PCI method and the kit method, both nDNA and mtDNA could be successfully detected in plasma samples, but nDNA extracted using protocol for single cell PCR was not detectable in 25 % of plasma samples. In comparison to the other two methods, the PCI method showed lower DNA purity, but higher concentrations and more GE of nDNA and mtDNA. Conclusions: The PCI method was more effi cient than the other two methods in the extraction of ccf DNA in plasma. Limited plasma is available for ccf DNA analysis.

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Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR

Cited by 32 publications

References 25 publications

Mitochondrial DNA Levels in Blood and Tissue Samples from Breast Cancer Patients of Different Stages

Mitochondrial DNA Levels in Blood and Tissue Samples from Breast Cancer Patients of Different Stages

Cell-free DNA in the urine of rats exposed to ionizing radiation

Strategies of reducing input sample volume for extracting circulating cell-free nuclear DNA and mitochondrial DNA in plasma

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