The art of obtaining a high yield of cell-free DNA from urine

Augustus, Elien; Casteren, Kaat Van; Sorber, Laure; Dam, Peter van; Roeyen, Geert; Peeters, Marc; Vorsters, Alex; Wouters, An; Raskin, Jo; Rolfo, Christian; Zwaenepoel, Karen; Pauwels, Patrick

doi:10.1371/journal.pone.0231058

Cited by 40 publications

(58 citation statements)

References 36 publications

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“…The collection of urine can be done in an outpatient setting or by self-sampling at home, and can easily be performed repeatedly. Moreover, urine appears to be a stable medium for the preservation of genetic material, when handled correctly [ 35 – 37 ]. This enables delivery to a testing laboratory per mail.…”

Section: Discussionmentioning

confidence: 99%

Non-invasive detection of endometrial cancer by DNA methylation analysis in urine

et al. 2020

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Background The incidence of endometrial cancer is rising, and current diagnostics often require invasive biopsy procedures. Urine may offer an alternative sample type, which is easily accessible and allows repetitive self-sampling at home. Here, we set out to investigate the feasibility of endometrial cancer detection in urine using DNA methylation analysis. Results Urine samples of endometrial cancer patients (n = 42) and healthy controls (n = 46) were separated into three fractions (full void urine, urine sediment, and urine supernatant) and tested for three DNA methylation markers (GHSR, SST, ZIC1). Strong to very strong correlations (r = 0.77–0.92) were found amongst the different urine fractions. All DNA methylation markers showed increased methylation levels in patients as compared to controls, in all urine fractions. The highest diagnostic potential for endometrial cancer detection in urine was found in full void urine, with area under the receiver operating characteristic curve values ranging from 0.86 to 0.95. Conclusions This feasibility study demonstrates, for the first time, that DNA methylation analysis in urine could provide a non-invasive alternative for the detection of endometrial cancer. Further investigation is warranted to validate its clinical usefulness. Potential applications of this diagnostic approach include the screening of asymptomatic women, triaging women with postmenopausal bleeding symptoms, and monitoring women with increased endometrial cancer risk.

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Section: Discussionmentioning

confidence: 99%

Non-invasive detection of endometrial cancer by DNA methylation analysis in urine

et al. 2020

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“…The volume range of ascitic fluid used in this study was in the range of 200 μl to 1 ml (depending on sample volume availability), and we believe that an increase in sample volume can ensure optimum amount of cfDNA extraction which will lead to fewer false negative results [43]. An improved sensitivity was obtained for urinary EGFR cfDNA detection in NSCLC patients when~90-100 ml of urine was used for analysis [44]. The specificity for the cfMTB-DNA qPCR assay was 97.1% (95% CI: 84.7,99.9) in both 'Definite and Probable' ATB group and combined 'ATB' group.…”

Section: Discussionmentioning

confidence: 99%

Utility of circulating cell-free Mycobacterium tuberculosis DNA for the improved diagnosis of abdominal tuberculosis

et al. 2020

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Abdominal tuberculosis (ATB) continues to pose a major diagnostic challenge for clinicians due to its nonspecific clinical presentation, variable anatomical location and lack of sensitive diagnostic tools. In spite of the development of several assays till date; no single test has proved to be adequate for ATB diagnosis. In this study, we for the first time report the detection of circulating cell-free Mycobacterium tuberculosis (M. tuberculosis) DNA (cfMTB-DNA) in ascitic fluid (AF) samples and its utility in ATB diagnosis. Sixty-five AF samples were included in the study and processed for liquid culture, cytological, biochemical and molecular assays. A composite reference standard (CRS) was formulated to categorize the patients into 'Definite ATB' (M. tuberculosis culture positive, n = 2), 'Probable ATB' (n = 16), 'Possible ATB' (n = 13) and 'Non-TB' category (n = 34). Two molecular assays were performed, namely, the novel cfMTB-DNA qPCR assay targeting M. tuberculosis devR gene and Xpert MTB/RIF assay (Xpert), and their diagnostic accuracy was assessed using CRS as reference standard. Clinical features such as fever, loss of weight, abdominal distension and positive Mantoux were found to be strongly associated with ATB disease (p<0.05). cfMTB-DNA qPCR had a sensitivity of 66.7% (95% CI:40.9,86.7) with 97.1% specificity (95% CI:84.7,99.9) in 'Definite ATB' and 'Probable ATB' group collectively. The sensitivity increased to 70.9% (95% CI:51.9,85.8) in the combined 'Definite', 'Probable' and 'Possible' ATB group with similar specificity. The cfMTB-DNA qPCR assay performed significantly better than the Xpert assay which demonstrated a poor sensitivity of �16.7% with 100% (95% CI:89.7,100) specificity (p<0.001). We conclude that cfMTB-DNA qPCR assay is an accurate molecular test that can provide direct evidence of M. tuberculosis etiology and has promise to pave the way for improving ATB diagnosis.

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“…Various commercial stabilization and preservation solutions were developed and used for cfNAs applications ( Table S3 ), including the Streck Cell-Free DNA Urine Preserve [ 147 ], Urine Conditioning Buffer [ 148 ], Norgen Urine Preservation [ 149 , 150 ], Colli Pee (DNA Genotek, Ottawa, ON, Canada) [ 151 ], or the Urine Collection Tube (Hunan UPSBio, Hunan, China) [ 136 ]. They are generally liquid reagents which can be added to the urine samples, or they may be available in a dried form in the collection tubes, to stabilize cfDNA and inhibit nuclease activity.…”

Section: Sample Collectionmentioning

confidence: 99%

Technical and Methodological Aspects of Cell-Free Nucleic Acids Analyzes

Pös

Styk

et al. 2020

IJMS

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Analyzes of cell-free nucleic acids (cfNAs) have shown huge potential in many biomedical applications, gradually entering several fields of research and everyday clinical care. Many biological properties of cfNAs can be informative to gain deeper insights into the function of the organism, such as their different types (DNA, RNAs) and subtypes (gDNA, mtDNA, bacterial DNA, miRNAs, etc.), forms (naked or vesicle bound NAs), fragmentation profiles, sequence composition, epigenetic modifications, and many others. On the other hand, the workflows of their analyzes comprise many important steps, from sample collection, storage and transportation, through extraction and laboratory analysis, up to bioinformatic analyzes and statistical evaluations, where each of these steps has the potential to affect the outcome and informational value of the performed analyzes. There are, however, no universal or standard protocols on how to exactly proceed when analyzing different cfNAs for different applications, at least according to our best knowledge. We decided therefore to prepare an overview of the available literature and products commercialized for cfNAs processing, in an attempt to summarize the benefits and limitations of the currently available approaches, devices, consumables, and protocols, together with various factors influencing the workflow, its processes, and outcomes.

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The art of obtaining a high yield of cell-free DNA from urine

Cited by 40 publications

References 36 publications

Non-invasive detection of endometrial cancer by DNA methylation analysis in urine

Non-invasive detection of endometrial cancer by DNA methylation analysis in urine

Utility of circulating cell-free Mycobacterium tuberculosis DNA for the improved diagnosis of abdominal tuberculosis

Technical and Methodological Aspects of Cell-Free Nucleic Acids Analyzes

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