Optimizing the yield and utility of circulating cell-free DNA from plasma and serum

Xue, Xiaoyan; Teare, M. Dawn; Holen, Ingunn; Zhu, Yimin; Woll, Penella J.

doi:10.1016/j.cca.2009.02.018

Cited by 138 publications

(122 citation statements)

References 25 publications

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“…In the samples collected from the two clinically normal dogs, there was an initial drop, followed by an increase in the cfDNA concentration, most notable when the whole blood was held at room temperature prior to separation. This dynamic process of continued cell death and DNA degradation ex vivo in samples prior to plasma isolation has also been found to occur in human samples and, similarly, is least marked when plasma is separated less than 2 h post sample collection (Jung et al 2003;Xue et al 2009). To minimize the impact of ex vivo variation in this study, all plasma was separated from blood samples in less than 2 h. However, once plasma has been separated from the cells, storage for 24 h prior to cfDNA analysis at 4 C did not cause a biologically relevant change in plasma cfDNA concentration.…”

Section: Discussionmentioning

confidence: 83%

“…The concentration of cfDNA has been measured in human plasma by various techniques like SYBR Gold fluorescence (Goldstein et al 2009), PicoGreen direct fluorescence and SYBR Green quantitative polymerase chain reaction (qPCR) (Xue et al 2009), and using the Qubit nucleic acid quantification system (Invitrogen 2010). Multiple ex vivo factors have been found to affect cfDNA stability, including storage temperature, time till plasma separation from cells and repeated freeze-thawing of the plasma (Jung et al 2003;Chan et al 2005).…”

Section: Introductionmentioning

confidence: 99%

“…Previously, DNA was isolated from the plasma samples and then amplified by polymerase chain reaction (PCR). However, this DNA extraction results in a loss of DNA from the sample (Goldstein et al 2009;Xue et al 2009;Jung et al 2010). The amount of DNA lost during extraction depends on the extraction technique but it can be as great as 80% (Xue et al 2009).…”

Section: Introductionmentioning

confidence: 99%

“…In addition, PCR is limited to the amplification of a specific sequence or fragment of the total cfDNA. More recently, studies have focused on nonspecific cfDNA quantification using emission fluorometry after the addition of a fluorochrome with specific affinity for DNA (Goldstein et al 2009;Xue et al 2009;Invitrogen 2010).…”

Section: Introductionmentioning

confidence: 99%

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Investigation of cell-free DNA in canine plasma and its relation to disease

Burnett

Cave

Gedye

et al. 2016

Veterinary Quarterly

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Background: DNA is released from dying cells during apoptosis and necrosis. This cell-free DNA (cfDNA) diffuses into the plasma where it can be measured. In humans, an increase in cfDNA correlates with disease severity and prognosis. Objective: It was hypothesized that when DNA in canine plasma was measured by emission fluorometry without prior DNA extraction, the concentration of cfDNA would increase with disease severity. Animals: The diseased population consisted of 97 client-owned dogs. The clinically normal population consisted of nine client-owned dogs presenting for 'wellness screens', and 15 colony-owned Harrier Hounds. Methods: Plasma cfDNA was measured by fluorometry without prior DNA extraction. The effects of ex vivo storage conditions were evaluated in plasma from two clinically normal dogs. In all other dogs, plasma was separated within two hours of collection. The association between the cfDNA concentration in hospitalized dogs and a variety of clinical, clinicopathological and outcome variables was tested. Results: The concentration of cfDNA was reliably measured when plasma was separated within two hours of blood collection. The diseased dogs had significantly higher cfDNA than clinically normal dogs (P < 0.001), and the more severe the disease, the higher the cfDNA when severity was categorized according to the American Society of Anesthesiologists (ASA) status (P < 0.001). Dogs that did not survive to discharge had significantly higher cfDNA concentrations than survivors (P D 0.02). Conclusions/Clinical Importance: The concentration of cfDNA in the plasma of diseased dogs is associated with disease severity and prognosis. Measurement of canine cfDNA could be a useful non-specific disease indicator and prognostic tool.

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Section: Discussionmentioning

confidence: 83%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Investigation of cell-free DNA in canine plasma and its relation to disease

Burnett

Cave

Gedye

et al. 2016

Veterinary Quarterly

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“…protocols according to Schmidt et al [8], Yuan et al [9], and Hufnagl et al [10], and a THP (triton/heat/phenol) protocol according to Xue et al [11].…”

Section: Extraction With Organic Solvents Eg Phenol-chloroform Sepamentioning

confidence: 99%

Isolation of Cell-Free DNA from Seminal Fluid

Draškovič¹

2017

JPP

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Cell-free DNA (cfDNA) is small short double stranded molecule that is also found in seminal fluid. It is a product of apoptotic cells in different developmental stages during spermatogenesis. To final concentration of total cfDNA in semen contributes also cfDNA secreted from living cells and cfDNA that is result of different diseases e.g. prostate cancer or infertility. Amended concentration (high or low) can be connected to prostate cancer or male infertility and can represent important non-invasive diagnostic biomarker for detection and prognosis of these pathological conditions. In this paper, I will discuss different approaches for isolation of cfDNA from seminal fluid, which includes selection of the samples, separation, isolation, extraction, purification and analysis. Today's most popular approach for isolation is the use of commercial kits based on selective binding and elution on silica-membrane technology, magnetic-bead technology or extraction with organic solvents and salting out procedure. Furthermore I will present that I tried to isolate cfDNA from semen with QIAamp DNA Mini Kit to confirm the presence of cell-free DNA in our samples. In the end I will describe problems we are facing during cfDNA measurement which are mainly associated with low concentration of cfDNA in samples.

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Application of circulating tumor DNA in prospective clinical oncology trials – standardization of preanalytical conditions

et al. 2017

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Circulating tumor DNA (ctDNA) has emerged as a potential new biomarker with diagnostic, predictive, and prognostic applications for various solid tumor types. Before beginning large prospective clinical trials to prove the added value of utilizing ctDNA in clinical practice, it is essential to investigate the effects of various preanalytical conditions on the quality of cell‐free DNA (cfDNA) in general and of ctDNA in particular in order to optimize and standardize these conditions. Whole blood samples were collected from patients with metastatic cancer bearing a known somatic variant. The following preanalytical conditions were investigated: (a) different time intervals to plasma isolation (1, 24, and 96 h) and (b) different preservatives in blood collection tubes (EDTA, CellSave, and BCT). The quality of cfDNA/ctDNA was assessed by DNA quantification, digital polymerase chain reaction (dPCR) for somatic variant detection and a β‐actin fragmentation assay for DNA contamination from lysed leukocytes. In 11 (69%) of our 16 patients, we were able to detect the known somatic variant in ctDNA. We observed a time‐dependent increase in cfDNA concentrations in EDTA tubes, which was positively correlated with an increase in wild‐type copy numbers and large DNA fragments (> 420 bp). Using different preservatives did not affect somatic variant detection ability, but did stabilize cfDNA concentrations over time. Variant allele frequency was affected by fluctuations in cfDNA concentration only in EDTA tubes at 96 h. Both CellSave and BCT tubes ensured optimal ctDNA quality in plasma processed within 96 h after blood collection for downstream somatic variant detection by dPCR.

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Optimizing the yield and utility of circulating cell-free DNA from plasma and serum

Abstract: Background. Cell-free DNA (CFDNA) in the plasma/serum of patients with cancer

Cited by 138 publications

References 25 publications

Investigation of cell-free DNA in canine plasma and its relation to disease

Investigation of cell-free DNA in canine plasma and its relation to disease

Isolation of Cell-Free DNA from Seminal Fluid

Application of circulating tumor DNA in prospective clinical oncology trials – standardization of preanalytical conditions

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