Analytical Comparison of Methods for Extraction of Short Cell-Free DNA from Urine

Oreskovic, Amy; Brault, Norman D.; Panpradist, Nuttada; Lai, James J.; Lutz, Barry

doi:10.1016/j.jmoldx.2019.07.002

Cited by 53 publications

(93 citation statements)

References 47 publications

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“…In addition, urinary cfDNA also reflects the systemic status of patients, as it is derived from both the urinary tract and the circulatory system [16]. However, although many studies have compared the various extraction methods of circulating cfDNA in blood [17][18][19], limited data are available on methods for urinary cfDNA extraction [20,21]. Most studies have been conducted on urinary DNA, regardless of being genomic DNA or cfDNA [22][23][24].…”

Section: Introductionmentioning

confidence: 99%

Comparison of Four Commercial Kits for Isolation of Urinary Cell-Free DNA and Sample Storage Conditions

Lee

Yoon

et al. 2020

Diagnostics

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Urinary cell-free DNA (cfDNA) is an attractive body fluid for liquid biopsy. In this study, we compared the efficiencies of four commercial kits for urinary cell-free DNA (cfDNA) isolation and of various sample storage conditions. Urinary cfDNA was isolated from 10 healthy individuals using four commercial kits: QIAamp Circulating Nucleic Acid Kit (QC; Qiagen), MagMAX™ Cell-Free DNA Isolation Kit (MM; Applied Biosystems), Urine Cell-Free Circulating DNA Purification Midi Kit (NU; Norgen Biotek), and Quick-DNA™ Urine Kit (ZQ; Zymo Research). To assess the isolation efficiency, an Agilent 2100 Bioanalyzer with High Sensitivity DNA chips was used, and cfDNA yield was defined as the amount of cfDNA obtained from 1 mL of urine. MM and QC provided the highest cfDNA yield in the 50–300 bp range, and MM and NU gave the highest cfDNA yield in the 50–100 bp range. In particular, the NU kit was efficient for isolation of more fragmented cfDNA in the range of 50–100 bp with the lowest cellular genomic DNA contamination. ZQ had the best cost-efficiency for isolating the same amount of urinary cfDNA. Samples stored at −70 °C with the addition of 10 mM EDTA resulted in the highest cfDNA yield 3 months after sample collection.

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Section: Introductionmentioning

confidence: 99%

Comparison of Four Commercial Kits for Isolation of Urinary Cell-Free DNA and Sample Storage Conditions

Lee

Yoon

et al. 2020

Diagnostics

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“…The copyright holder for this preprint this version posted January 25, 2021. ; https://doi.org/10.1101/2021.01.19.21249296 doi: medRxiv preprint 21 binding of short cfDNA, but did not report its analytical performance (e.g., percent recovery) (8). While the Wizard method improves upon conventional silica-based methods, our past testing revealed limited recovery (<35%) of ≤150 bp fragments (31). It was also highly dependent on urine composition and is likely to fail in samples with high pH and/or low nontarget DNA concentrations (31) Past studies also enrolled few smear-negative participants, and thus have not tested the ability to detect urine cfDNA in individuals with paucibacillary TB who stand to benefit most from a non-sputum-based test.…”

Section: Discussionmentioning

confidence: 93%

“…While the Wizard method improves upon conventional silica-based methods, our past testing revealed limited recovery (<35%) of ≤150 bp fragments (31). It was also highly dependent on urine composition and is likely to fail in samples with high pH and/or low nontarget DNA concentrations (31) Past studies also enrolled few smear-negative participants, and thus have not tested the ability to detect urine cfDNA in individuals with paucibacillary TB who stand to benefit most from a non-sputum-based test. In particular, the most promising study by Cannas et al included only 5% (2/43) smear-negative participants.…”

Section: Discussionmentioning

confidence: 93%

Diagnosing pulmonary tuberculosis using sequence-specific purification of urine cell-free DNA

Oreskovic¹,

Panpradist²,

Marangu³

et al. 2021

Preprint

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Transrenal urine cell-free DNA (cfDNA) is a promising tuberculosis (TB) biomarker, but is challenging to detect because of the short length (<100 bp) and low concentration of TB-specific fragments. We aimed to improve the diagnostic sensitivity of TB urine cfDNA by increasing recovery of short fragments during sample preparation. We developed a highly sensitive sequence-specific purification method that uses hybridization probes immobilized on magnetic beads to capture short TB cfDNA (50 bp) with 91.8% average efficiency. Combined with short-target PCR, the assay limit of detection was ≤5 copies of cfDNA in 10 mL urine. In a clinical cohort study in South Africa, our urine cfDNA assay had 83.7% sensitivity (95% CI: 71.0– 91.5%) and 100% specificity (95% CI: 86.2–100%) for diagnosis of active pulmonary TB when using sputum Xpert MTB/RIF as the reference standard. The detected cfDNA concentration was 0.14–2804 copies/mL (median 14.6 copies/mL) and was inversely correlated with CD4 count and days to culture positivity. Sensitivity was non-significantly higher in HIV-positive (88.2%) compared to HIV-negative patients (73.3%), and was not dependent on CD4 count. Sensitivity remained high in sputum smear-negative (76.0%) and urine LAM-negative (76.5%) patients. With improved sample preparation, urine cfDNA is a viable biomarker for TB diagnosis. Our assay has the highest reported accuracy of any TB urine cfDNA test to date and has the potential to enable rapid non-sputum-based TB diagnosis across key underserved patient populations.

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“…Alternatively, colorimetric quantitation is used, and more sensitive in the sub‐µg range 21 . A different approach utilizes quantitative real‐time PCR for DNA quantification, of which the reproducibility is however dependent on the initial DNA extraction method chosen, as well as potential interference from non‐DNA components in the sample itself 12,22 …”

Section: Resultsmentioning

confidence: 99%

“…Especially for sfDNA, however, this does not recover the total amount. Investigations into the recovery of sfDNA from solid‐phase extraction kits have shown that for DNA fragments < 50 bp and < 100 bp, only about 16.5% and 27.7% (median across various extraction kits) are recovered, respectively 11‐13 …”

Section: Introductionmentioning

confidence: 99%

Solid‐phase silica‐based extraction leads to underestimation of residual DNA in decellularized tissues

Schmitz

Eren

Spierings

et al. 2020

Xenotransplantation

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Decellularization of animal tissues is a novel route to obtain biomaterials for use in tissue engineering and organ transplantation. Successful decellularization is required as animal DNA causes inflammatory reactions and contains endogenous retroviruses, which could be transmitted to the patient. One of the criteria for successful decellularization is digestion (fragmentation) and elimination (residual quantity) of DNA from the tissue. Quantification of DNA can be done in many ways, but it has recently been shown that silica‐based solid‐phase extraction methods often do not completely purify in particular small DNA fragments. In the context of decellularization, this means that the measured DNA amount is underestimated, which could compromise safety of the processed tissue for in‐patient use. In this article, we review DNA quantification methods used by researchers and assess their influence on the reported DNA contents after decellularization. We find that underestimation of residual DNA amount after silica‐based solid‐phase extraction may be as large as a factor of ten. We therefore recommend a direct assessment of DNA amount in tissue lysate using dsDNA‐specific binding dyes, such as Picogreen, due to their higher accuracy for small fragment detection as well as ease of use and widespread availability.

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Analytical Comparison of Methods for Extraction of Short Cell-Free DNA from Urine

Cited by 53 publications

References 47 publications

Comparison of Four Commercial Kits for Isolation of Urinary Cell-Free DNA and Sample Storage Conditions

Comparison of Four Commercial Kits for Isolation of Urinary Cell-Free DNA and Sample Storage Conditions

Diagnosing pulmonary tuberculosis using sequence-specific purification of urine cell-free DNA

Solid‐phase silica‐based extraction leads to underestimation of residual DNA in decellularized tissues

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