New method to preserve the original proportion and integrity of urinary cell‐free <scp>DNA</scp>

Li, Pei; Ning, Jun; Luo, Xipeng; Du, Hongwu; Zhang, Qing; Zhou, Ganlin; Du, Qian; Ou, Zhenyu; Wang, Long; Yu, Wei

doi:10.1002/jcla.22668

Cited by 15 publications

(16 citation statements)

References 36 publications

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“…Similar results, i.e. no change in the total amount of DNA in urine supernatant from bladder cancer patients, which had been stored for 7 days at RT in containers containing a proprietary preservative, were reported by Li et al [191].…”

Section: Choice Of Matrix -Plasma Vs Serum Vs Other Body Fluidssupporting

confidence: 88%

Pre-analytical issues in liquid biopsy – where do we stand?

Fleischhacker

Schmidt

2020

Journal of Laboratory Medicine

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It is well documented that in the chain from sample to the result in a clinical laboratory, the pre-analytical phase is the weakest and most vulnerable link. This also holds for the use and analysis of extracellular nucleic acids. In this short review, we will summarize and critically evaluate the most important steps of the pre-analytical phase, i.e. the choice of the best control population for the patients to be analyzed, the actual blood draw, the choice of tubes for blood drawing, the impact of delayed processing of blood samples, the best method for getting rid of cells and debris, the choice of matrix, i.e. plasma vs. serum vs. other body fluids, and the impact of long-term storage of cell-free liquids on the outcome. Even if the analysis of cell-free nucleic acids has already become a routine application in the area of non-invasive prenatal screening (NIPS) and in the care of cancer patients (search for resistance mutations in the EGFR gene), there are still many unresolved issues of the pre-analytical phase which need to be urgently tackled.

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Section: Choice Of Matrix -Plasma Vs Serum Vs Other Body Fluidssupporting

confidence: 88%

Pre-analytical issues in liquid biopsy – where do we stand?

Fleischhacker

Schmidt

2020

Journal of Laboratory Medicine

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“…Therefore, inhibition of DNases might be crucial to prevent cfDNA degradation. One of the effective ways is to use treatment with EDTA and store urine samples at −70 °C or below for a long-time or to use sterile containers and store samples at 4 °C or on ice and process them as soon as possible [ 136 ]. Despite such conditions, the loss of cfDNA may occur [ 137 ].…”

Section: Sample Collectionmentioning

confidence: 99%

“…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%

“…Other single step protocols were also reported to be functional, such as single centrifugation at 4000× g for 10 min, or 3000× g for 15 min [ 147 ], suggesting a wide functional range for centrifugation protocols. On the other hand, there are studies that prefer a double centrifugation protocol, first at 200× g followed by 1800× g for 10 min [ 153 ], or first low-speed centrifugation at 1600× g for 10 min followed by second high-speed centrifugation at 16,000× g for 10 min at 4 °C [ 136 ]. Two-step centrifugation at 200× g for 10 min followed by a second centrifugation at 1000× g for 10 min was also reported to be used [ 152 ].…”

Section: Processing and Storage Of Biological Materialsmentioning

confidence: 99%

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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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“…cfDNA was extracted from plasma using the QIAamp circulating nucleic acid kit (QIAGEN) according to the manufacturer's instructions. cfDNA from clinical samples and mice was quantified using a single locus qPCR assay on the beta-actin (ACTB / Actb) gene [33,34]. Fragment analysis of cfDNA was performed using Agilent DNA Bioanalyser.…”

Section: Genomic and Cell-free Dna Extraction And Quantitationmentioning

confidence: 99%

Absolute measurement of the tissue origins of cell-free DNA in the healthy state and following paracetamol overdose

et al. 2020

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Background: Despite the emergence of cell-free DNA (cfDNA) as a clinical biomarker in cancer, the tissue origins of cfDNA in healthy individuals have to date been inferred only by indirect and relative measurement methods, such as tissue-specific methylation and nucleosomal profiling. Methods: We performed the first direct, absolute measurement of the tissue origins of cfDNA, using tissue-specific knockout mouse strains, in both healthy mice and following paracetamol (APAP) overdose. We then investigated the utility of total cfDNA and the percentage of liver-specific cfDNA as clinical biomarkers in patients presenting with APAP overdose. Results: Analysis of cfDNA from healthy tissue-specific knockout mice showed that cfDNA originates predominantly from white and red blood cell lineages, with minor contribution from hepatocytes, and no detectable contribution from skeletal and cardiac muscle. Following APAP overdose in mice, total plasma cfDNA and the percentage fraction originating from hepatocytes increased by~100 and~19-fold respectively. Total cfDNA increased by an average of more than 236-fold in clinical samples from APAP overdose patients with biochemical evidence of liver injury, and 18-fold in patients without biochemically apparent liver injury. Measurement of liver-specific cfDNA, using droplet digital PCR and methylation analysis, revealed that the contribution of liver to cfDNA was increased by an average of 175-fold in APAP overdose patients with biochemically apparent liver injury compared to healthy subjects, but was not increased in overdose patients with normal liver function tests. Conclusions: We present a novel method for measurement of the tissue origins of cfDNA in healthy and disease states and demonstrate the potential of cfDNA as a clinical biomarker in APAP overdose.

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New method to preserve the original proportion and integrity of urinary cell‐free DNA

Cited by 15 publications

References 36 publications

Pre-analytical issues in liquid biopsy – where do we stand?

Pre-analytical issues in liquid biopsy – where do we stand?

Technical and Methodological Aspects of Cell-Free Nucleic Acids Analyzes

Absolute measurement of the tissue origins of cell-free DNA in the healthy state and following paracetamol overdose

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