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Gentle, Effective, and Timely Isolation of Extracellular Vesicles that is Amenable for Transcriptomics Using Next-Generation Aqueous Two-Phase System Technology

ABSTRACT: Gentle, Effective, and Timely Isolation of Extracellular Vesicles that is Amenable for Transcriptomics Using Next-Generation Aqueous Two-Phase System Technology

PROVIDER: PRJNA1120025 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR29281400.fastq.gz	Fastqsanger.gz
	SRR29281401.fastq.gz	Fastqsanger.gz
	SRR29281402.fastq.gz	Fastqsanger.gz
	SRR29281403.fastq.gz	Fastqsanger.gz
	SRR29281404.fastq.gz	Fastqsanger.gz

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Gentle, Effective, and Timely Isolation of Extracellular Vesicles that is Amenable for Transcriptomics Using Next-Generation Aqueous Two-Phase System Technology

Project description:The isolation of extracellular vesicles (EVs) using currently available methods frequently compromises purity and yield to prioritize speed. Here, we present a next generation aqueous two-phase system (ATPS) for isolation of EVs regardless of scale and volume and is superior to conventional methods such as ultracentrifugation and commercial kits. This is made possible by the two aqueous phases, one rich in polyethylene glycol (PEG), and the other rich in dextran (DEX), whereby fully encapsulated lipid vesicles preferentially migrate to the DEX-rich phase to achieve a local energy minimum for the EVs. Isolated EVs as found in the DEX-rich phase are more amenable to biomarker analysis such as nanoscale flow cytometry when using various pre-conjugated antibodies specific for CD9, CD63, and CD81. TRIzol RNA isolation is further enabled by the addition of Dextranase, a critical component of this next generation ATPS method. This negates the use of specialized EV RNA extraction kits. The use of Dextranase also enables more accurate immunoreactivity of pre-conjugated antibodies for detection of EVs by nanoscale flow cytometry. Transcriptomics of isolated EVs with ATPS revealed strong overlap between micro RNA (miRNA), circular RNA (circRNA), and small nucleolar RNA (snoRNA) profiles of EVs isolated compared to UC and was superior to other kits. Overall, this ATPS method stands out as a rapid and highly effective approach to isolate EVs, ensuring optimal extraction and analysis of nucleic acids.

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Next Generation Aqueous Two-Phase System for Gentle, Effective, and Timely Extracellular Vesicle Isolation and Transcriptomic Analysis.

Project description:The isolation of extracellular vesicles (EVs) using currently available methods frequently compromises purity and yield to prioritize speed. Here, we present a next-generation aqueous two-phase system (next-gen ATPS) for the isolation of EVs regardless of scale and volume that is superior to conventional methods such as ultracentrifugation (UC) and commercial kits. This is made possible by the two aqueous phases, one rich in polyethylene glycol (PEG) and the other rich in dextran (DEX), whereby fully encapsulated lipid vesicles preferentially migrate to the DEX-rich phase to achieve a local energy minimum for the EVs. Isolated EVs as found in the DEX-rich phase are more amenable to biomarker analysis such as nanoscale flow cytometry (nFC) when using various pre-conjugated antibodies specific for CD9, CD63 and CD81. TRIzol RNA isolation is further enabled by the addition of dextranase, a critical component of this next-gen ATPS method. RNA yield of next-gen ATPS-isolated EVs is superior to UC and other commercial kits. This negates the use of specialized EV RNA extraction kits. The use of dextranase also enables more accurate immunoreactivity of pre-conjugated antibodies for the detection of EVs by nFC. Transcriptomic analysis of EVs isolated using the next-gen ATPS revealed a strong overlap in microRNA (miRNA), circular RNA (circRNA) and small nucleolar RNA (snoRNA) profiles with EV donor cells, as well as EVs isolated by UC and the exoRNeasy kit, while detecting a superior number of circRNAs compared to the kit in human samples. Overall, this next-gen ATPS method stands out as a rapid and highly effective approach to isolate high-quality EVs in high yield, ensuring optimal extraction and analysis of EV-encapsulated nucleic acids.

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Project description:Purpose: To characterize microRNAs (miRNAs) and their possible roles in high myopia by using next generation sequencing Methods: Aqueous humor samples were obtained from 15 highly myopic eyes and 15 cataract eyes at the onset of surgery. miRNA next generation sequencing and bioinformatics analyses were performed using RNA extracted from aqueous humor samples. Results: A total of 341 miRNAs were detected in the aqueous humor samples of highly myopic eyes; 201 miRNAs were detected in the aqueous humor samples of cataractous control eyes. A total of 249 mature miRNAs and 17 novel miRNAs were differentially expressed during myopia. Possible pathways regulated by these aberrantly expressed miRNAs included the TNF, MAPK, PI3K-Akt, and HIF-1 signaling pathways. Conclusions: The current study provided an overall view of miRNA profiling in the aqueous humor of highly myopic eyes. These profiles may be associated with myopia pathogenesis, and are potential biomarkers.

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Organotypic human bronchial epithelial cultures (MucilAir) exposed to aqueous aerosol extracts of the 3R4F cigarette smoke and a novel tobacco product vapor

Project description:The current study was performed for analysis of the biological effects of vapor from novel tobacco vapor product in comparison with 3R4F cigarette smoke. This study was performed using a three-dimensional culture system composed of an air-liquid interface culture of primary normal human bronchial epithelial cells (MucilAir). The MucilAir tissues were subjected to 17 days of exposure to the aqueous extract of novel tobacco product vapor or 3R4F cigarette smoke. The number of differentially expressed genes increased in MucilAir tissues exposed to aqueous extract of each test product dependent on exposure duration. The number of differentially expressed genes was lower in the tissues exposed to aqueous extract of novel tobacco product vapor compared to the tissues exposed to aqueous extract of 3R4F cigarette smoke.

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