Project description:RI-SEC-seq: small RNA sequencing in size-exclusion chromatography fractions of MCF-7 cell-conditioned medium treated with/without RNase inhibitor
Project description:Extracellular vesicles (EVs) released by human placenta–derived mesenchymal stem cells (hpMSCs) are thought to mediate paracrine effects. To characterize their small-RNA cargo, we isolated hpMSC-derived EVs (hpEVs) from conditioned media using size-exclusion chromatography and performed miRNA sequencing.
Project description:The current study is focused on Isolating EVs and their cargo content (miRNAs) from culture medium conditioned by individual embryos that can differentiate between the blastocyst and non-blastocyst embryos. We were able to isolate EVs from both blastocyst and non-blastocyst group with size exclusion chromatography (Izon’s™ qEV single column) and characterize them with Western blotting, nanoparticle tracking analysis, and Transmission electron microscopy. Further, we could isolate total RNA (including small RNAs) from the blastocyst and non-blastocyst EVs for miRNA sequencing.
Project description:The human amniotic membrane (hAM) is a valuable tissue in regenerative medicine due to its capacity to release cell-derived bioactive factors. Among these, microRNAs (miRNAs) play a critical role in modulating gene expression. This study aimed to comprehensively characterize the miRNA landscape of native viable hAM tissue and its secretome, with a focus on distinguishing between extracellular vesicles (EV)-associated and protein-bound miRNAs. We analyzed two anatomically and functionally distinct regions of the hAM, the reflected and placental amnion. After incubation of tissue biopsies ex vivo for 72 hours, vesicular and non-vesicular components in the conditioned medium were separated by size exclusion chromatography, and small RNA sequencing was performed on tissue and secretome fractions. Our analysis identified three main clusters of miRNA expression corresponding to tissue, EV-associated, and protein-bound fractions. We observed regional differences in miRNA expression between the reflected and placental amnion and identified miRNAs selectively released into EVs or protein-bound fractions. Data of gene ontology analysis suggest distinct biological roles for miRNA depending on the sample type. This dataset provides novel insights into the spatial and functional miRNA release profile of viable hAM and contributes to a better understanding of its regenerative potential.
Project description:Plasma was harvested from two cohorts of facility-matched germ free (GF) and specific-pathogen free (SPF) mice at the National Gnotobiotic Rodent Resource Center (NGRRC; North Carolina, USA). Plasma was then fractionated by size-exclusion chromatography using three tandem Superdex200 increase columns (Cytiva). Fractions corresponding with HDL were then pooled and concentrated prior to RNA isolation. Small RNA libraries were generated from total RNA using NextFlex V3 Small RNA Seq-kit (Perkin Elmer) according to manufacturer’s instructions. Equimolar amounts of each library were then pooled and sequenced on the NextSeq500 platform (Illumina). Individual libraries were then demultiplexed and analyzed with the TIGER analytical pipeline.
Project description:To evaluate performance of immunomagnetic separation and size exclusion chromatography in the isolation of different extracellular RNA carriers from biofluids.
Project description:Plasma from lung cancer patients from EDTA tubes was fractionated using size exclusion chromatography. Fractions 1-5, 7-11, 12-15, 16-20 were pooled, cfDNA was extracted from the fractions and paired unfractionated samples and PE150bp sequencing was performed on an Illumina Novaseq S4 flowcell. Samples are provided as raw reads without any prior processing.
Project description:We studied extracellular vesicles (EVs) from synovial fibroblast (SF). EVs were isolated from the secretome of non-senescent and irradiation-induced senescent SFs using size exclusion chromatography. EV RNA was extracted and subject to small RNA sequencing on an Illumina NovaSeq SP using 100bp, single end reads. After mapping against GRCh38.p12 and miRBase v22.1 differential expression analysis was undertaken with edgeRv3.28 using a quasi-likelihood negative binomial generalized log-linear model. We identified a panel of differentially expressed small non-coding RNAs.
Project description:Experiment aimed at understanding the compositional changes of small extracellular vesicles (sEVs) derived from cells grown under hyperthermic conditions. Human embryonic kidney (HEK293) cells were cultured 48 h under normal conditions (37°C/140 rpm/5% CO2) before being subjected to a 72 h, 40°C temperature shift. sEVs were then isolated from the supernatant via tangential flow filtration (TFF) and size exclusion chromatography (SEC)