Optimization of Extracellular Small RNA Isolation Methods for Transcriptomic Profiling of Urinary Extracellular Vesicles in Pregnancy

Placenta ◽  
2021 ◽  
Vol 112 ◽  
pp. e31-e32
Author(s):  
Priyadarshini Pantham ◽  
Srimeenakshi Srinivasan ◽  
Peter DeHoff ◽  
Aishwarya Vuppala ◽  
Amber Morey ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Small Rna ◽  
Rna Isolation ◽  
Transcriptomic Profiling ◽  
Isolation Methods ◽  
In Pregnancy

scholarly journals Comprehensive evaluation of extracellular small RNA isolation methods from serum in high throughput sequencing

BMC Genomics ◽  
2017 ◽  
Vol 18 (1) ◽  
Author(s):  
Yan Guo ◽  
Kasey Vickers ◽  
Yanhua Xiong ◽  
Shilin Zhao ◽  
Quanhu Sheng ◽  
...  
Keyword(s):  
High Throughput ◽  
Small Rna ◽  
High Throughput Sequencing ◽  
Comprehensive Evaluation ◽  
Rna Isolation ◽  
Isolation Methods

scholarly journals Enhanced recovery of CD9-positive extracellular vesicles from human specimens by chelating reagent

2020 ◽  
Author(s):  
Ayako Kurimoto ◽  
Yuki Kawasaki ◽  
Toshiki Ueda ◽  
Tatsutoshi Inuzuka
Keyword(s):  
Extracellular Vesicles ◽  
Western Blotting ◽  
Biological Samples ◽  
Liquid Biopsy ◽  
Enhanced Recovery ◽  
Recovery Efficiency ◽  
Early Diagnostics ◽  
Isolation Methods ◽  
Chelating Reagent

AbstractExtracellular vesicles (EVs) have gained attention as potential targets of early diagnostics and prognosis in the field of liquid biopsy. Despite clinical potentials, the best method to isolate EVs from specimens remains controversial due to low purity, low specificity, and lack of reproducibility with current isolation methods. Here we show that a chelating reagent enhances the recovery efficiency of EVs from crude biological samples by immunoprecipitation using an anti-CD9 antibody. Proteomic and western blotting analyses show that the EVs isolated using the chelating reagent contain a wider variety of proteins than those isolated with PBS.

scholarly journals Transcriptomic profiling of the oyster pathogen Vibrio splendidus opens a window on the evolutionary dynamics of the small RNA repertoire in the Vibrio genus

RNA ◽  
2012 ◽  
Vol 18 (12) ◽  
pp. 2201-2219 ◽  
Author(s):  
C. Toffano-Nioche ◽  
A. N. Nguyen ◽  
C. Kuchly ◽  
A. Ott ◽  
D. Gautheret ◽  
...  
Keyword(s):  
Small Rna ◽  
Evolutionary Dynamics ◽  
Vibrio Splendidus ◽  
Transcriptomic Profiling ◽  
Vibrio Genus

88 MicroRNA profile of invitro bovine embryos cultured in the presence of amniotic extracellular vesicles shifts toward invivo-collected blastocysts

2020 ◽  
Vol 32 (2) ◽  
pp. 170
Author(s):  
A. Lange-Consiglio ◽  
B. Lazzari ◽  
F. Pizzi ◽  
A. Idda ◽  
F. Cremonesi ◽  
...  
Keyword(s):  
Pregnancy Rate ◽  
Extracellular Vesicles ◽  
Embryo Implantation ◽  
Principal Component ◽  
Rna Isolation ◽  
Differentially Expressed ◽  
Bovine Embryos ◽  
Total Rna ◽  
Mirna Profiling ◽  
Genomic Study

The absence of maternal-embryo signals could be an important cause of the poor pregnancy rates of invitro-produced embryos, compared with those collected invivo. In the context of paracrine communication, co-culture of embryo with amniotic-derived extracellular vesicles (EVs) improved their quality compared with control (CTR) (Perrini and Lange Consiglio 2018 Reprod. Fertil. Dev. 30, 658-671), and after cryopreservation, provided higher invitro embryo hatching and recipient pregnancy rate (Lange-Consiglio et al. 2019 Reprod. Fertil. Dev. 31, 155). After these results, the aim of this study was to evaluate microRNA (miRNA) profiling of invitro-produced blastocysts with or without EV supplementation, using invivo-produced blastocysts as CTR. Invitro embryos were produced based on our protocol (Perrini and Lange Consiglio 2018 Reprod. Fertil. Dev. 30, 658-671) with or without 100×106 EVsmL−1 in synthetic oviductal fluid with amino acids (SOFaa) on Day 5 post-fertilisation (Perrini and Lange Consiglio 2018 Reprod. Fertil. Dev. 30, 658-671). Grade 1 blastocysts (B7) were immediately snap frozen in liquid nitrogen for genomic study. These embryos were obtained from three replicates. Invivo embryos were obtained from three cows superovulated by Folltropin and inseminated by the same cryopreserved semen. After flushing, only B7 were snap frozen for genomic study. Samples for RNA isolation were obtained from 3 pools of 10 embryos each for each condition (vivo, vitro-CTR, and vitro+EVs). Total RNA was isolated by a NucleoSpin1 miRNA kit. Concentration and quality of RNA were determined by an Agilent 2100 Bioanalyzer. Libraries were prepared using TruSeq Small RNA Library Preparation kits (Illumina). Differential expression analyses between samples were run with the Bioconductor edgeR package (false discovery rate<0.05). MicroRNA cluster analysis was performed with Genesis. The average quantity of total RNA extracted from each pool was 3.5ng. Our results show that the miRNAs identified were 1.74E5, 2.3E5, and 3.6E5 for vivo, vitro-CTR, and vitro+EVs, respectively. Principal component analysis calculated on differentially expressed miRNAs showed a separation of the three groups with a distinctive miRNA trait. The miRNAs differentially expressed among three comparisons (vivo vs. vitro-CTR, vivo vs. vitro+EVs, and vitro-CTR vs. vitro+EVs) were 20, 15, and 2, respectively. Principal component 1, which explains 62.4% of the variance, clearly separates invivo- and invitro-produced embryos even if EV addition seems to ameliorate the effect of invitro production, and this agrees with the embryo quality and pregnancy rate after EV supplementation (Perrini and Lange Consiglio 2018 Reprod. Fertil. Dev. 30, 658-671; Lange-Consiglio et al. 2019 Reprod. Fertil. Dev. 31, 155). Indeed, vitro-CTR and vitro+EVs embryos differ significantly for two miRNAs (miR-130a, miR-181b) that are found to be higher in our vitro-CTR embryos compared with vitro+EV ones. The miR-181b was also found to be higher in degenerate bovine embryos compared with good blastocysts (Kropp et al. 2014 Front. Genetics 24, 91). In conclusion, this is the first study reporting the complete miRNA profiling of invitro blastocysts compared with those obtained invivo. The addition of EVs during invitro production seems to influence the expression of specific miRNAs involved in the success of embryo implantation.

scholarly journals Emerging role of extracellular vesicles in the regulation of skeletal muscle adaptation

2019 ◽  
Vol 127 (2) ◽  
pp. 645-653 ◽  
Author(s):  
Ivan J. Vechetti
Keyword(s):  
Skeletal Muscle ◽  
Extracellular Vesicles ◽  
Human Body ◽  
Intercellular Communication ◽  
Skeletal Muscle Mass ◽  
Genetic Material ◽  
Muscle Adaptation ◽  
Pathological Conditions ◽  
Isolation Methods

Extracellular vesicles (EVs) were initially characterized as “garbage bags” with the purpose of removing unwanted material from cells. It is now becoming clear that EVs mediate intercellular communication between distant cells through a transfer of genetic material, a process important to the systemic adaptation in physiological and pathological conditions. Although speculative, it has been suggested that the majority of EVs that make it into the bloodstream would be coming from skeletal muscle, since it is one of the largest organs in the human body. Although it is well established that skeletal muscle secretes peptides (currently known as myokines) into the bloodstream, the notion that skeletal muscle releases EVs is in its infancy. Besides intercellular communication and systemic adaptation, EV release could represent the mechanism by which muscle adapts to certain stimuli. This review summarizes the current understanding of EV biology and biogenesis and current isolation methods and briefly discusses the possible role EVs have in regulating skeletal muscle mass.

scholarly journals 3D cell culture stimulates the secretion of in vivo like extracellular vesicles

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Sirisha Thippabhotla ◽  
Cuncong Zhong ◽  
Mei He
Keyword(s):  
Cervical Cancer ◽  
Cell Culture ◽  
Cancer Patient ◽  
Extracellular Vesicles ◽  
Small Rna ◽  
Small Rnas ◽  
3D Culture ◽  
Cervical Cancer Patient ◽  
Tumor Biomarker

Abstract For studying cellular communications ex-vivo, a two-dimensional (2D) cell culture model is currently used as the “gold standard”. 2D culture models are also widely used in the study of RNA expression profiles from tumor cells secreted extracellular vesicles (EVs) for tumor biomarker discovery. Although the 2D culture system is simple and easily accessible, the culture environment is unable to represent in vivo extracellular matrix (ECM) microenvironment. Our study observed that 2D- culture derived EVs showed significantly different profiles in terms of secretion dynamics and essential signaling molecular contents (RNAs and DNAs), when compared to the three-dimensional (3D) culture derived EVs. By performing small RNA next-generation sequencing (NGS) analysis of cervical cancer cells and their EVs compared with cervical cancer patient plasma EV-derived small RNAs, we observed that 3D- culture derived EV small RNAs differ from their parent cell small RNA profile which may indicate a specific sorting process. Most importantly, the 3D- culture derived EV small RNA profile exhibited a much higher similarity (~96%) to in vivo circulating EVs derived from cervical cancer patient plasma. However, 2D- culture derived EV small RNA profile correlated better with only their parent cells cultured in 2D. On the other hand, DNA sequencing analysis suggests that culture and growth conditions do not affect the genomic information carried by EV secretion. This work also suggests that tackling EV molecular alterations secreted into interstitial fluids can provide an alternative, non-invasive approach for investigating 3D tissue behaviors at the molecular precision. This work could serve as a foundation for building precise models employed in mimicking in vivo tissue system with EVs as the molecular indicators or transporters. Such models could be used for investigating tumor biomarkers, drug screening, and understanding tumor progression and metastasis.

scholarly journals Micro Ribonucleic Acid Profiles of Traumatic Brain Injury Isolated From Plasma Extracellular Vesicles

Neurosurgery ◽  
2019 ◽  
Vol 66 (Supplement_1) ◽  
Author(s):  
Ross Puffer ◽  
Luz Cumba-Garcia ◽  
Benjamin T Himes ◽  
David O Okonkwo ◽  
Ian F Parney
Keyword(s):  
Traumatic Brain Injury ◽  
Brain Injury ◽  
Extracellular Vesicles ◽  
Rna Isolation ◽  
Extracellular Vesicle ◽  
Micro Rna ◽  
Altered Consciousness ◽  
Rna Sequences ◽  
Altered Level ◽  
Level Of Consciousness

Abstract INTRODUCTION Extracellular vesicles (EVs) are membrane-bound particles released by the majority of human cells, including cells within the central nervous system. They may represent a diagnostic or prognostic target obtainable in peripheral blood of neurotrauma patients. We have isolated micro RNA sequences contained within EVs of 15 patients with traumatic brain injury (TBI) and compared them to miRNA sequences from 5 healthy controls. METHODS Extracellular vesicles were isolated from 15 TBI subjects, including 6 mild TBI (Glasgow Coma Scale (GCS) 13-15), 3 moderate TBI (GCS 9-12), and 6 severe TBI (GCS 3-8), as well as 5 healthy control. EVs were analyzed using nanoparticle tracking analysis. Samples underwent RNA isolation and extraction, followed by miRNA sequencing and analysis. RESULTS TBI patients presenting with an altered level of consciousness (GCS = 14) had a significantly higher mean extracellular vesicle size compared to subjects with normal GCS (mean + /− sem = 108.3 nm + /− 7.7 nm vs 89.2 nm + /− 6.7 nm; P < .04). GFAP ELISA of the samples demonstrated significantly higher GFAP concentration in subjects with altered level of consciousness (GCS = 14) as compared to those with normal GCS (mean + /− sem GFAP concentration 2204.2 pg/mL + /− 1067.2 pg/mL vs. 207.8 pg/mL + /− 270.8 pg/mL, P = .05). We identified 9 miRNA sequences that were found in a significantly higher proportion in patients with altered consciousness compared to controls, as well as 2 miRNA sequences that were significantly downregulated in subjects with altered consciousness as compared to controls. CONCLUSION EVs may contain brain specific biomarkers that are released in greater quantities after TBI. These molecules can be isolated from plasma and sequenced. Further analysis will better elucidate the final pathways affected by these up and downregulated miRNA sequences. Analysis of EVs in subjects with TBI may allow for the identification of novel diagnostic and potentially prognostic biomarkers.

scholarly journals INSIDE Project: Individual Air Pollution Exposure, Extracellular Vesicles Signaling and Hypertensive Disorder Development in Pregnancy

2020 ◽  
Vol 17 (23) ◽  
pp. 9046
Author(s):  
Luca Ferrari ◽  
Francesca Borghi ◽  
Simona Iodice ◽  
Dolores Catelan ◽  
Stefano Rossi ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Pregnancy Outcomes ◽  
Molecular Mechanisms ◽  
Screening Program ◽  
Health Impact ◽  
Hypertensive Disorder ◽  
Geostatistical Models ◽  
Study Population ◽  
Pollution Exposure ◽  
In Pregnancy

Hypertensive disorders are common complications during pregnancy (HDP) with substantial public health impact. Acute and chronic particulate matter (PM) exposure during pregnancy increases the risk of HDP, although the underlying molecular mechanisms remain unclear. Extracellular vesicles (EVs) may be the ideal candidates for mediating the effects of PM exposure in pregnancy as they are released in response to environmental stimuli. The INSIDE project aims to investigate this mechanism in pregnancy outcomes. The study population is enrolled at the Fetal Medicine Unit of Fondazione IRCCS Ca’Granda—Ospedale Maggiore Policlinico at 10–14 weeks of gestation. Exposure to PM10 and PM2.5 is assessed using the flexible air quality regional model (FARM) and Bayesian geostatistical models. Each woman provides a blood sample for EV analysis and circulating biomarker assessment. Moreover, a subgroup of recruited women (n = 85) is asked to participate in a cardiovascular screening program including a standard clinical evaluation, a non-invasive assessment of right ventricular function, and pulmonary circulation at rest and during exercise. These subjects are also asked to wear a personal particulate sampler, to measure PM10, PM2.5, and PM1. The INSIDE study is expected to identify the health impacts of PM exposure on pregnancy outcomes.

scholarly journals Comparison of Total RNA Isolation Methods for Analysis of Immune-Related microRNAs in Market Milks

2015 ◽  
Vol 35 (4) ◽  
pp. 459-465 ◽  
Author(s):  
Sangnam Oh ◽  
Mi Ri Park ◽  
Seok Jun Son ◽  
Younghoon Kim
Keyword(s):  
Rna Isolation ◽  
Total Rna ◽  
Isolation Methods ◽  
Total Rna Isolation
Sign in / Sign up

Export Citation Format

Share Document