Obtaining High-Quality Blood Specimens for Downstream Applications: A Review of Current Knowledge and Best Practices

Li, Qiyuan; Xian, Wang; Li, Xue; He, Xuheng; Wan, Qian; Yin, Jiefang; Sun, Jianbo; Yang, Xiaoping; Qiao-hong, Chen; Miao, Xinyuan

doi:10.1089/bio.2018.0052

Cited by 5 publications

(3 citation statements)

References 78 publications

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“…When designing experiments for the discovery of biomarkers and profiling of EV components, it becomes essential to have a sample size that gives a statistical power of at least 80% [53]. Finally, information regarding the age, sex, race, disease state, and treatment state of the patient/animal should be collected to understand the impact of each of these parameters on the profiling of the EV content [54].…”

Section: Sample Collection Sample Matching Sample Size and Data Collectionmentioning

confidence: 99%

Technologies and Standardization in Research on Extracellular Vesicles

Gandham

Wood

et al. 2020

Trends in Biotechnology

340

285

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Despite the substantial recent progress made in extracellular vesicle (EV) research, our understanding of the functional and mechanistic biology of EVs and their relevance to specific pathophysiological states remains limited.Detailed characterization of the molecular composition of EVs and EV subpopulations remains a challenge.Alternative, similar, or identical experimental approaches may often lead to substantially different EV profiling results in different laboratories.Standard protocols for specimen procurement, collection, preprocessing, EV isolation, analytical characterization, and data analysis/interpretation need to be developed for specialized applications and analytical workflows, optimized, documented, cross-evaluated by several laboratories, and disseminated to further accelerate progress toward further understanding of EV biology and development of novel EV-based diagnostic and therapeutic approaches.

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Section: Sample Collection Sample Matching Sample Size and Data Collectionmentioning

confidence: 99%

Technologies and Standardization in Research on Extracellular Vesicles

Gandham

Wood

et al. 2020

Trends in Biotechnology

340

285

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“…2 Efforts performed in the field have been focused on isolating these vesicles from plasma and serum as these body fluids are abundant, easily accessible, and routinely collected through minimally invasive procedures. 9,10 However, the use of blood-derived EVs in a clinical context depends on the capacity to isolate these structures from contaminants in sufficient yields and in a reproducible, cost-effective, and simple manner. [11][12][13][14][15] Among the different techniques available so far, size exclusion chromatography (SEC) holds great promise for EVbased translational research as this method can be easily adapted to most research and clinical laboratories.…”

Section: Introductionmentioning

confidence: 99%

Simple and Fast SEC-Based Protocol to Isolate Human Plasma-Derived Extracellular Vesicles for Transcriptional Research

Gaspar

Santana

Henriques

et al. 2020

Molecular Therapy - Methods & Clinical Development

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Extracellular vesicles (EVs) are membranous structures that protect RNAs from damage when circulating in complex biological fluids, such as plasma. RNAs are extremely specific to health and disease, being powerful tools for diagnosis, treatment response monitoring, and development of new therapeutic strategies for several diseases. In this context, EVs are potential sources of disease biomarkers and promising delivery vehicles. However, standardized and reproducible EV isolation protocols easy to implement in clinical practice are missing. Here, a size exclusion chromatography-based protocol for EV-isolation from human plasma was optimized. We propose a workflow to isolate EVs for transcriptional research that allows concomitant analysis of particle number and size, total protein, and quantification of a major plasma contaminant. This protocol yields 7.54 × 10 9 ± 1.22 × 10 8 particles, quantified by nanoparticle tracking analysis, with a mean size of 115.7 ± 11.12 nm and a mode size of 83.13 ± 4.72 nm, in a ratio of 1.19 × 10 10 ± 7.38 × 10 9 particles/μg of protein, determined by Micro Bicinchoninic Acid (BCA) Protein Assay, and 3.09 ± 0.7 ng RNA, assessed by fluorescence-based RNA-quantitation, from only 900 μL of plasma. The protocol is fast and easy to implement and has potential for application in biomarkers research, therapeutic strategies development, and clinical practice.

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“…Sin embargo, a pesar de estos valores no óptimos (proteínas residuales y contaminantes traza) obtenidos en los cinco métodos de extracción de ADN, no se observó inhibición de las reacciones enzimáticas por PCR. Aplicaciones moleculares más sensibles tales como secuenciación Sanger, genotipado PCR-RFLP, microarreglos o NGS, podrían verse afectadas por la presencia de sales, solventes orgánicos, EDTA, nucleasas y proteínas contaminantes que se acarrean en los ADN aislados (21)(22)(23)(24) . Los bajos valores obtenidos con el coeficiente A260/A230 en muestras extraídas a partir de los kits comerciales pudo deberse a compuestos con absorción a 230 nm actuando como contaminantes traza, los cuales incluyen sales caotrópicas como el tiocianato de guanidina (25) , EDTA, detergentes no iónicos como Triton™ X-100 y Tween®, proteínas, aminoácidos (20,25) , fenol, polisacáridos y otras partículas sólidas flotantes como las fibras de sílice.…”

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Incubación, pre-lisis y post-purificación en el rendimiento y pureza de ácidos nucleicos extraídos de sangre de cabras domésticas contenida en tarjetas FTA

Sancho-Blanco

Jiménez-Alfaro

Molina‐Bravo

et al. 2022

Rev. Mex. Cienc. Pecu.

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Técnicas moleculares requieren extracciones de ácidos nucleicos en cantidad y pureza adecuadas. Este trabajo describe un modelo lineal generalizado (GLM) de un factor ajustado con efectos fijos sobre el rendimiento de ácido nucleico (ng/μl) y la pureza (A260/A280 y A260/A230), para cinco métodos de extracción de ADN utilizando tarjetas FTA con sangre de cabra (Capra aegagrus hircus). Se ensayaron dos métodos comerciales basados en columnas de sílice (Invitrogen y Macherey Nagel; MN), método resina quelante (Chelex), método CTAB y el método de fenol-cloroformo-alcohol isoamílico (PCI). Adicionalmente, para MN, se evaluó una etapa de incubación con tampón PBS (Phosphate Buffered Saline) en alta temperatura previa a la lisis y una etapa de purificación posterior a la extracción utilizando un modelo de efecto fijo de dos factores con interacción. Las concentraciones de ADN y las proporciones de pureza fueron variables; la concentración más alta se obtuvo con el kit MN (170.45 ng/μl), pero con deficiencias en la pureza (0.32 de A260/A230, 0.34 de A260/A280). A pesar de esto, todos los métodos de extracción generaron productos PCR con cebadores específicos D-loop (ADNmt). El efecto combinado de las etapas de pre-incubación y post-purificación arrojó valores de pureza satisfactorios (1.89 para A260/A230 y 1.65 para A260/A280), así como relaciones de concentración (476.78 ng/μl) con baja variabilidad. En conclusión, la concentración y pureza del ADN de muestras de sangre mejora considerablemente cuando se usa un kit comercial en combinación con incubación previa a la lisis y purificación posterior a la extracción. Estos ácidos nucleicos se sugieren para uso en potenciales aplicaciones moleculares a posteriori.

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Obtaining High-Quality Blood Specimens for Downstream Applications: A Review of Current Knowledge and Best Practices

Cited by 5 publications

References 78 publications

Technologies and Standardization in Research on Extracellular Vesicles

Technologies and Standardization in Research on Extracellular Vesicles

Simple and Fast SEC-Based Protocol to Isolate Human Plasma-Derived Extracellular Vesicles for Transcriptional Research

Incubación, pre-lisis y post-purificación en el rendimiento y pureza de ácidos nucleicos extraídos de sangre de cabras domésticas contenida en tarjetas FTA

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