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.

Technical and Methodological Aspects of Cell-Free Nucleic Acids Analyzes

Gaining a better understanding of the biological properties of cell-free DNA constitutes an important step in the development of clinically meaningful cell-free DNA-based tests. Since the in vivo characterization of cell-free DNA is complicated by the immense heterogeneity of blood samples, an increasing number of in vitro cell culture experiments, which offer a greater level of control, are being conducted. However, cell culture studies are currently faced with three notable caveats. First, the concentration of cell-free DNA in vitro is relatively low. Second, the median amount and size of cell-free DNA in culture medium varies greatly between cell types. Third, the amount and size of cell-free DNA in the culture medium of a single cell line fluctuates over time. Although these are interesting findings, it can also be a great source of experimental confusion and emphasizes the importance of method optimization and standardization. Therefore, in this study, we compared five commonly used cell-free DNA quantification methods, including quantitative polymerase chain reaction, Qubit Double-Stranded DNA High Sensitivity assay, Quant-iT PicoGreen Assay, Bioanalyzer High Sensitivity DNA assay, and NanoDrop Onec. Analysis of the resulting data, along with an interpretation of theoretical values (i.e. the theoretical detection and quantification limits of the respective methods), enables the calculation of optimal conditions for several important preanalytical steps pertaining to each quantification method and different cell types, including the (1) time-point at which culture medium should be collected for cell-free DNA extraction, (2) amount of cell culture supernatant from which to isolate cell-free DNA, (3) volume of elution buffer, and (4) volume of cell-free DNA sample to use for quantification.

https://journals.sagepub.com/doi/pdf/10.1177/1010428319866369

Comparison of methods for the quantification of cell-free DNA isolated from cell culture supernatant:

Abstract 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.

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

Cancer biomarkers are a promising tool for cancer detection, personalization of therapy, and monitoring of treatment response or recurrence. “Liquid biopsy” commonly refers to minimally invasive or non-invasive sampling of a bodily fluid (i.e., blood, urine, saliva) for detection of cancer biomarkers such as circulating tumor cells or cell-free tumor DNA (ctDNA). These methods offer a means to collect frequent tumor assessments without needing surgical biopsies. Despite much progress with blood-based liquid biopsy approaches, there are limitations—including the limited amount of blood that can be drawn from a person and challenges with collecting blood samples at frequent intervals to capture ctDNA biomarker kinetics. These limitations are important because ctDNA is present at extremely low levels in plasma and there is evidence that measuring ctDNA biomarker kinetics over time can be useful for clinical prediction. Additionally, blood-based assays require access to trained phlebotomists and often a trip to a healthcare facility. In contrast, urine is a body fluid that can be self-collected from a patient’s home, at frequent intervals, and mailed to a laboratory for analysis. Multiple reports indicate that fragments of ctDNA pass from the bloodstream through the kidney’s glomerular filtration system into the urine, where they are known as trans-renal ctDNA (TR-ctDNA). Accumulating studies indicate that the limitations of blood based ctDNA approaches for cancer can be overcome by measuring TR-ctDNA. Here, we review current knowledge about TR-ctDNA in urine as a cancer biomarker approach, and discuss its clinical potential and open questions in this research field.

/pdf/trans-renal-cell-free-tumor-dna-for-urine-based-liquid-3sczmpr1.pdf

Trans-Renal Cell-Free Tumor DNA for Urine-Based Liquid Biopsy of Cancer

Liquid biopsy, through isolation and analysis of disease-specific analytes, has evolved as a promising tool for safe and minimally invasive diagnosis and monitoring of tumors. It also has tremendous utility as a companion diagnostic allowing detection of biomarkers in a range of cancers (lung, breast, colon, ovarian, brain). However, clinical implementation and validation remains a challenge. Among other stages of development, preanalytical variables are critical in influencing the downstream cellular and molecular analysis of different analytes. Although considerable progress has been made to address these challenges, a comprehensive assessment of the impact on diagnostic parameters and consensus on standardized and optimized protocols is still lacking. Here, we summarize and critically evaluate key variables in the preanalytical stage, including study population selection, choice of biofluid, sample handling and collection, processing, and storage. There is an unmet need to develop and implement comprehensive preanalytical guidelines on the optimal practices and methodologies. 

Extrinsic and intrinsic preanalytical variables affecting liquid biopsy in cancer.

The invention relates to a nucleic acid purification reagent based on a magnetic bead method and a kit. The nucleic acid purification reagent is prepared from a water solution containing the followingsubstances in percentage by mass: 1 to 5 percent of carboxyl-modified magnetic beads, 11.78 to 23.56 percent of sodium chloride, 10 to 20 percent of polyethylene glycol, 0.01 to 0.05 percent of N-sodium lauroyl sarcosine, 0.16 to 0.79 percent of Tris-HCl, 0.5 to 2 percent of EDTA and 0.5 to 2 percent of tween-20. A nucleic acid purification recovery kit comprises the nucleic acid purification reagent, a washing buffer solution and an elution buffer solution. The nucleic acid purification reagent and the kit have the advantages that the nucleic acid recovery efficiency is higher than the recovery rate of the existing magnetic bead type reagent and kit; the recovery loss on trace samples is lower; the recovery efficiency on nucleic acid fragments being about 100bp or smaller nucleic acid fragments is better.

Nucleic acid purification reagent based on magnetic bead method

A urine free DNA extraction kit comprise lysate, detergent A, detergent B, eluent and hydroxyl magnetic beads, wherein that lysate comprise guanidinium salt, metal chelating agent, surfactant, alcohol. Tris-HCl and sodium chloride, detergent A include guanidinium salts, metal chelate agents, surfactants, ethanol and tris-HCl, ethanol, 30%-40% by volume. The detergent B includes metal chelating agent, surfactant, ethanol and Tris-HCl. The volume concentration of HCl and ethanol is 75% -85%. The invention also provides a method for extracting urine free DNA by using the kit. The kit provided bythe invention has high extraction efficiency for free DNA in urine and selectively extracts nucleic acid fragments of more than 100 bp.

A urine free DNA extraction kit and an extraction method thereof

The invention relates to a kit for detecting a bladder cancer based on a high-throughput sequencing technique. The kit comprises bladder cancer mutant gene multiple PCR (polymerase chain reaction) specific primers shown from SEQ ID NO.1 to SEQ ID NO.64 and joint elements. According to the kit, a plurality of bladder cancer mutant gene loci can be simultaneously amplified through urine nucleic acid, so that a sequencing library is built, bladder cancer risks of detected people are analyzed through high-throughput sequencing and biological information, the kit is convenient in sampling, high inaccuracy and noninvasive, and the pain of a patient is relieved.

Kit for detecting bladder cancer based on high-throughput sequencing technique

The invention relates to a kit for prenatal non-invasive paternity testing. The kit comprises a part of or all the primers in a primer set for detecting 86 SNP loci. The kit of the invention can realize prenatal non-invasive paternity testing by using maximal 86 SNP loci. The kit and a detection method provided by the invention can realize high-sensitivity, high-throughput and low-cost detection,and can obtain sequences with an average depth of 20, 000 by using only 5 M of data; and with the kit and the detection method, non-invasive early-stage detection of kinship can be realized before thebirth of a child, the accuracy of the detection exceeds 99.99%, and families with very close kinship can also be perfectly distinguished.

Kit and method for prenatal non-invasive paternity testing

Background Due to high nuclease activity and complex contents in urine, urinary cell-free DNA (ucfDNA) was prone to degrade. So, we developed standardized urine collection tube (UCT) to prevent ucfDNA degradation and simultaneously maintain urinary cells in their original form during the sample collection process, ensuring stabilization of the original proportion and integrity of ucfDNA. Methods Urine samples were collected from bladder cancer patients and divided into 10-mL normal tubes and 10-mL UCTs, respectively, and kept at ambient temperature. Urine supernatant was separated by centrifuging, and ucfDNA was extracted. Then ucfDNA was quantified by quantitative real-time polymerase chain reaction. UcfDNA fragments distribution was analyzed by Agilent 2200, and the frequency of specific mutations of urinary system disease was detected by next-generation sequencing method. Results Urine collected into UCTs showed no statistically significant changes in their original proportion and integrity of ucfDNA up to 7 days at ambient temperature and also ucfDNA fragments were maintained well. Conversely, urine collected into normal tubes was observed an obviously decline in their original proportion of ucfDNA and ucfDNA fragments changed greatly. The △% of allele fraction (AF) for specific genes of ucfDNA from UCTs was lower than from normal tubes by 3.7-fold. Conclusion Using UCTs, they can maximally keep the original proportion and integrity of ucfDNA and stabilize urinary cells and minimize the background noise caused by urinary cellular DNA releasing, it will be help to open the door of next-generation noninvasive liquid biopsy applications utilizing urine.

/pdf/new-method-to-preserve-the-original-proportion-and-integrity-szyux5hdaf.pdf

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

Handle everyday research tasks with reliable, citation-backed results

Your personal Research Agent to handle research tasks with citation-backed results

Popular Tasks used by Researchers

How can I help with your research?

Meet SciSpace

Get more enhanced response by uploading the PDFs you want me to reference.

No relevant PDFs in your library

SciSpace is the AI research assistant for academics. Run systematic literature reviews on 280M+ papers, and write papers with cited sources. Trusted by 1M+ students, PhDs & researchers.

SciSpace | AI for Research

Analyze PDFs

Code & Manuscripts

Funding & Grants

Literature & Patents

Medical & Clinical Data

Systematic Review

Visualize & Present

Web & Data

Build a Google Scholar-like website for your research.

Build a website

Create charts and images for your research

Create a Chart

Write a paper for submission to a journal

Draft a manuscript

Patent Search

Design eye-catching scientific posters in minutes.

Scientific Poster Generation

Systematic Literature Review

One task is running at the moment. Your messages will be shown right after.

Drag and drop or click here to browse

Loved by <highlight>1 million+</highlight> researchers

Extract a list of specific topics and their sources from unstructured text

Topics

Compare and analyze relevant papers that matches with your search

Papers

Get insights from PDFs and bookmarked papers from your library

My library

Recent searches

Try searching for:

Catch AI-generated content in scholarly and non-scholarly content

{ai} Detector

Ai Writer

Get PDF Summaries, highlighted text explanations 

Chat with PDF

Effortlessly create in-text citations and bibliographies in APA and 2,500 other formats

Citation generator

Get explanations, summaries, and answers on academic papers

Ease up your research workflow with {scispace}'s cohort of exciting AI tools

Elevate your academic writing skills and convey your ideas the way you want

Paraphraser

Explore our range of reading and writing tools

Your file is being prepared and should be ready in a few minutes. If it's a large file, it might take a bit longer. You can close this window, and we'll email you the file when it's done.

You have reached a maximum limit of <strong>{limit}</strong> columns in the table. Remove at least <strong>1</strong> column to add or create another one.

Luo Xipeng

Author Tools

Chat about Author