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Dye-free spectrophotometric measurement of nucleic acid-to-protein ratio for cell-selective extracellular vesicle discrimination.

ABSTRACT: Extracellular vesicles (EVs) can represent a novel source of disease biomarkers, and are under intensive study for their clinical potential. Most EV-based cancer diagnostic studies have focused on establishing EV assays that detect increased expression of a single cancer-associated marker or marker signatures based on multiplex detection of these biomarkers. EV biomarker readouts can be obscured by high background signal leading to false positives, and may markedly differ between analyses due to variation in sample purity during EV isolation. This can obstruct the comparisons among studies and lead to conflicting conclusions. This work reports that the nucleic acid to protein UV absorption ratio of an EV is a cell-specific EV characteristic. This EV collective attribute can be measured at low-cost to discriminate EVs derived from malignant and non-malignant cells rather than employing single markers that may be cancer- or subtype-specific. Our work also highlighted the application for accessing purity in EV preparations irrelevant to EV yield. It can be employed to distinguish from patients with and without malignant disease upon analysis of EVs isolated from their serum samples.

SUBMITTER: Sun D

PROVIDER: S-EPMC7995647 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature

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Dye-free spectrophotometric measurement of nucleic acid-to-protein ratio for cell-selective extracellular vesicle discrimination.

Sun Dali D Zhao Zhen Z Spiegel Sarah S Liu Yang Y Fan Jia J Amrollahi Pouya P Hu Jing J Lyon Christopher J CJ Wan Meihua M Hu Tony Y TY

Biosensors & bioelectronics 20210209

Extracellular vesicles (EVs) can represent a novel source of disease biomarkers, and are under intensive study for their clinical potential. Most EV-based cancer diagnostic studies have focused on establishing EV assays that detect increased expression of a single cancer-associated marker or marker signatures based on multiplex detection of these biomarkers. EV biomarker readouts can be obscured by high background signal leading to false positives, and may markedly differ between analyses due to ...[more]

PMID: 33592557

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Project description:BackgroundThe human sweat is a mixture of secretions from three types of glands: eccrine, apocrine, and sebaceous. Eccrine glands open directly on the skin surface and produce high amounts of water-based fluid in response to heat, emotion, and physical activity, whereas the other glands produce oily fluids and waxy sebum. While most body fluids have been shown to contain nucleic acids, both as ribonucleoprotein complexes and associated with extracellular vesicles (EVs), these have not been investigated in sweat. In this study we aimed to explore and characterize the nucleic acids associated with sweat particles.ResultsWe used next generation sequencing (NGS) to characterize DNA and RNA in pooled and individual samples of EV-enriched sweat collected from volunteers performing rigorous exercise. In all sequenced samples, we identified DNA originating from all human chromosomes, but only the mitochondrial chromosome was highly represented with 100% coverage. Most of the DNA mapped to unannotated regions of the human genome with some regions highly represented in all samples. Approximately 5 % of the reads were found to map to other genomes: including bacteria (83%), archaea (3%), and virus (13%), identified bacteria species were consistent with those commonly colonizing the human upper body and arm skin. Small RNA-seq from EV-enriched pooled sweat RNA resulted in 74% of the trimmed reads mapped to the human genome, with 29% corresponding to unannotated regions. Over 70% of the RNA reads mapping to an annotated region were tRNA, while misc. RNA (18,5%), protein coding RNA (5%) and miRNA (1,85%) were much less represented. RNA-seq from individually processed EV-enriched sweat collection generally resulted in fewer percentage of reads mapping to the human genome (7-45%), with 50-60% of those reads mapping to unannotated region of the genome and 30-55% being tRNAs, and lower percentage of reads being rRNA, LincRNA, misc. RNA, and protein coding RNA.ConclusionsOur data demonstrates that sweat, as all other body fluids, contains a wealth of nucleic acids, including DNA and RNA of human and microbial origin, opening a possibility to investigate sweat as a source for biomarkers for specific health parameters.

Project description:Ocean acidification, a phenomenon of seawater pH decrease due to increasing atmospheric CO2, has a global effect on seawater chemistry, marine biology, and ecosystems. Ocean acidification is a gradual and global long-term process, the study of which demands high-quality pH data. The spectrophotometric technique is capable of generating accurate and precise pH measurements but requires adding an indicator dye that perturbs the sample original pH. While the perturbation is modest in well-buffered seawater, applications of the method in environments with lower buffer capacity such as riverine, estuarine, sea-ice meltwater and lacustrine environments are increasingly common, and uncertainties related to larger potential dye perturbations need further evaluation. In this paper, we assess the effect of purified meta-Cresol Purple (mCP) dye addition on the sample pH and how to correct for this dye perturbation. We conducted numerical simulations by incorporating mCP speciation into the MATLAB CO2SYS program to examine the changes in water sample pH caused by the dye addition and to reveal the dye perturbation mechanisms. Then, laboratory experiments were carried out to verify the simulation results. The simulations suggest that the dye perturbation on sample pH is a result of total alkalinity (TA) contributions from the indicator dye and chemical equilibrium shifts that are related to both the water sample properties (pH, TA, and salinity) and the indicator dye solution properties (pH and solvent matrix). The laboratory experiments supported the simulation results; the same dye solution can lead to different dye perturbations in water samples with different pH, TA, and salinity values. The modeled adjustments agreed well with the empirically determined adjustments for salinities > 5, but it showed greater errors for lower salinities with disagreements as large as 0.005 pH units. Adjustments are minimized when the pH and salinity of the dye are matched to the sample. When the dye is used over a wide range of salinity, we suggest that it should be prepared in deionized water to minimize the dye perturbation effect on pH in the fresher sample waters with less well-constrained perturbation adjustments. We also suggest that the dye perturbation correction should be based on double dye addition experiments performed over a wide range of pH, TA, and salinity. Otherwise, multiple volume dye addition experiments are recommended for each sample to determine the dye perturbation adjustment. We further create a MATLAB function dyeperturbation.m that calculates the expected dye perturbation. This function can be used to validate empirically-derived adjustments or in lieu of empirical adjustments if dye addition experiments are unfeasible (e.g., for historical data). This study of dye perturbation evaluation and correction will improve the accuracy of the pH data, necessary for monitoring the long-term anthropogenic-driven changes in the seawater carbonate system.

Dataset Information

Dye-free spectrophotometric measurement of nucleic acid-to-protein ratio for cell-selective extracellular vesicle discrimination.

Publications

Dye-free spectrophotometric measurement of nucleic acid-to-protein ratio for cell-selective extracellular vesicle discrimination.

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