scholarly journals Urinary extracellular vesicles for RNA extraction: optimization of a protocol devoid of prokaryote contamination

2016 ◽  
Vol 5 (1) ◽  
pp. 30281 ◽  
Author(s):  
Dorota Tataruch-Weinert ◽  
Luca Musante ◽  
Oliver Kretz ◽  
Harry Holthofer
Keyword(s):  
Extracellular Vesicles ◽  
Rna Extraction ◽  
Extraction Optimization

36 Analysis of miRNA content of oviduct and uterine extracellular vesicles across the bovine estrous cycle

2021 ◽  
Vol 33 (2) ◽  
pp. 125
Author(s):  
M. Hamdi ◽  
R. Mazzarella ◽  
K. Cañon-Beltrán ◽  
Y. N. Cajas ◽  
C. L. V. Leal ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Target Genes ◽  
Embryo Implantation ◽  
Rna Extraction ◽  
Bioinformatic Analysis ◽  
Progressive Increase ◽  
Biological Pathways ◽  
Oestrous Cycle ◽  
Size Exclusion ◽  
Mirna Content

With the aim of investigating the possible hormonal regulatory effect of the oestrous cycle on miRNA content in the extracellular vesicles (EVs) of bovine oviducal and uterine fluids (OF, UF), we performed a bioinformatic analysis of these miRNAs, their target genes, and their biological pathways. Reproductive tracts were collected from slaughtered heifers and selected according to their corpus luteum morphology, corresponding to the 4 stages of the oestrous cycle (n=5 per stage; S1: days 1 to 4, S2: days 5–10, S3: days 11–17, S4: days 18–20) and transported to the laboratory on ice. EVs were obtained by size exclusion chromatography (PURE-EVs-Hansa Biomed) from the flushing of 1.2mL and 2.5mL of OF and UF, respectively. To concentrate the EVs, they were ultracentrifuged and suspended in 100µL of PBS. Total RNA extraction was obtained from 70µL of the previous pellet, using miRNeasy Mini Kit (Qiagen). Then, 100 to 200ng of the obtained RNA was reverse transcribed using miScript II RT Kit (Qiagen). MicroRNA (miRNA) expression profiling was done by primer-based real-time quantitative PCR of 383 mature miRNA sequences. Possible miRNA target genes and their biological pathways were predicted using the miRWalk database. Among EV miRNAs in OF, bta-miR-130a, bta-miR-382, and bta-miR-1291 were the most abundant at all stages of the oestrous cycle, displaying a significantly progressive increase from stages 1 to 4 (P<0.05). In UF, bta-miR-17-5p, bta-miR-206, bta-miR-22-5p, bta-miR-502a, and bta-miR-503-3p were the most abundant at all stages of the cycle, showing greater differences between S1 and S3 (P<0.05). Other miRNAs were exclusively present in a specific stage of the oestrous cycle in OF: bta-miR-21-5p (S1), bta-miR-146a (S2), bta-miR-128 (S3), and bta-miR-147 (S4). In UF, bta-miR-218 (S1), bta-miR-208b (S2), bta-miR-340 (S3), and bta-miR-335 (S4) were found. Table 1 presents some of these miRNAs, their predicted target genes, and functional pathways. In conclusion, this study highlights the effect of the oestrous cycle on miRNAs contained in the EVs of OF and UF. These miRNAs are related to relevant biological pathways implicated in oviduct and uterus modulation across the cycle, but they may also prepare those organs for embryo/conceptus presence and development. Table 1. Micro (mi)RNAs of oviductal (OF) and uterine fluid (UF) extracellular vesicles (EVs), their target genes, and biological pathways Reproductive fluid miRNAs Target genes Target pathways OF bta-miR-130a BMPR2, SMAD5, SMAD4 BMP signalling bta-miR-1291 SLC2A1 Glucagon signalling bta-miR-21–5p LIF Pluripotency stem cells regulation UF bta-miR-17-5p STAT3 Prolactin signalling bta-miR-206 ESR1 Oestrogen signalling bta-miR-340 HRAS Ras/MAPK/ERK signalling (embryo implantation) This research was funded by MINECO-Spain AGL2015-70140-R, PID2019-111641RB-I00, RTI2018-093548-B-I00; SENESCYT-Ecuador (YNC); FAPESP-Brazil 2017/20339-3 (CLVL), 2014/22887-0 (JCS), 2019/04981-2 (RM); CNPq-Brazil 304276/2018-9, 420152/2018-0 (CLVL).

scholarly journals Putative Internal Control Genes in Bovine Milk Small Extracellular Vesicles Suitable for Normalization in Quantitative Real Time-Polymerase Chain Reaction

Membranes ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 933
Author(s):  
Md. Matiur Rahman ◽  
Shigeo Takashima ◽  
Yuji O. Kamatari ◽  
Yassien Badr ◽  
Kaori Shimizu ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Extracellular Vesicles ◽  
Internal Control ◽  
Rna Extraction ◽  
Bovine Milk ◽  
Chain Reaction ◽  
Qrt Pcr ◽  
Internal Control Genes ◽  
Polymerase Chain

Bovine milk small extracellular vesicles (sEVs) contain many biologically important molecules, including mRNAs. Quantitative real-time polymerase chain reaction (qRT-PCR) is a widely used method for quantifying mRNA in tissues and cells. However, the use, selection, and stability of suitable putative internal control genes in bovine milk sEVs for normalization in qRT-PCR have not yet been identified. Thus, the aim of the present study was to determine suitable putative internal control genes in milk sEVs for the normalization of qRT-PCR data. Milk sEVs were isolated from six healthy Holstein-Friesian cattle, followed by RNA extraction and cDNA synthesis. In total, 17 mRNAs were selected for investigation and quantification using qRT-PCR; they were further evaluated using geNorm, NormFinder, BestKeeper, and ∆CT algorithms to identify those that were highly stable putative internal control genes in milk sEVs. The final ranking of suitable putative internal control genes was determined using RefFinder. The mRNAs from TUB, ACTB, DGKZ, ETFDH, YWHAZ, STATH, DCAF11, and EGFLAM were detected in milk sEVs from six cattle by qRT-PCR. RefFinder demonstrated that TUB, ETFDH, and ACTB were highly stable in milk sEVs, and thus suitable for normalization of qRT-PCR data. The present study suggests that the use of these genes as putative internal control genes may further enhance the robustness of qRT-PCR in bovine milk sEVs. Since these putative internal control genes apply to healthy bovines, it would be helpful to include that the genes were stable in sEVs under “normal or healthy conditions”.

scholarly journals High-Throughput and Automated Acoustic Trapping of Extracellular Vesicles to Identify microRNAs With Diagnostic Potential for Prostate Cancer

2021 ◽  
Vol 11 ◽  
Author(s):  
Anson Ku ◽  
Jacob Fredsøe ◽  
Karina D. Sørensen ◽  
Michael Borre ◽  
Mikael Evander ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Rna Sequencing ◽  
Extracellular Vesicles ◽  
Small Rna ◽  
Rna Extraction ◽  
Clinical Samples ◽  
Small Rna Sequencing ◽  
High Grade ◽  
Enrichment Technique ◽  
Diagnostic Potential

Molecular profiling of extracellular vesicles (EVs) offers novel opportunities for diagnostic applications, but the current major obstacle for clinical translation is the lack of efficient, robust, and reproducible isolation methods. To bridge that gap, we developed a microfluidic, non-contact, and low-input volume compatible acoustic trapping technology for EV isolation that enabled downstream small RNA sequencing. In the current study, we have further automated the acoustic microfluidics-based EV enrichment technique that enables us to serially process 32 clinical samples per run. We utilized the system to enrich EVs from urine collected as the first morning void from 207 men referred to 10-core prostate biopsy performed the same day. Using automated acoustic trapping, we successfully enriched EVs from 199/207 samples (96%). After RNA extraction, size selection, and library preparation, a total of 173/199 samples (87%) provided sufficient materials for next-generation sequencing that generated an average of 2 × 106 reads per sample mapping to the human reference genome. The predominant RNA species identified were fragments of long RNAs such as protein coding and retained introns, whereas small RNAs such as microRNAs (miRNA) accounted for less than 1% of the reads suggesting that partially degraded long RNAs out-competed miRNAs during sequencing. We found that the expression of six miRNAs was significantly different (Padj < 0.05) in EVs isolated from patients found to have high grade prostate cancer [ISUP 2005 Grade Group (GG) 4 or higher] compared to those with GG3 or lower, including those with no evidence of prostate cancer at biopsy. These included miR-23b-3p, miR-27a-3p, and miR-27b-3p showing higher expression in patients with GG4 or high grade prostate cancer, whereas miR-1-3p, miR-10a-5p, and miR-423-3p had lower expression in the GG4 PCa cases. Cross referencing our differentially expressed miRNAs to two large prostate cancer datasets revealed that the putative tumor suppressors miR-1, miR-23b, and miR-27a are consistently deregulated in prostate cancer. Taken together, this is the first time that our automated microfluidic EV enrichment technique has been found to be capable of enriching EVs on a large scale from 900 μl of urine for small RNA sequencing in a robust and disease discriminatory manner.

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