Small RNA sequencing of cerebrospinal fluid exosome fraction in medulloblastoma patients
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE157916 | GEO | 2020/09/15
REPOSITORIES: GEO
Similar Datasets
Project description:This series represents a group of five samples (from sigmoid colon biopsies of clinically normal Patients A-E) profiled by a publicly available cDNA-based platform (GPL284) and a commercial oligonucleotide-based platform (GPL91). Sample RNA extraction: Biopsies taken during endoscopic analysis were immediately snap-frozen in liquid nitrogen and at no time prior to or during subsequent sample manipulation were the samples allowed to thaw. Standard laboratory procedures to eliminate the presence of RNases, including the use of RNase free plastic-ware, heat baked glassware, molecular biology grade chemicals and DEPC-treated solutions, were adopted. Frozen biopsies were crushed to a fine powder under liquid nitrogen using a manual crusher with a teflon head (Omnilab) and total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturers protocol, with minor modifications that improved RNA yields and purity in our hands. In brief, crushed biopsy samples were lysed in an appropriate volume of Buffer RLT (typically 600 µL/20 mg tissue). To facilitate complete lysis, samples were allowed to stand at room temperature for 5-10 min before vortexing. The lysate was homogenized by addition to a QIA shredder column (Qiagen) and centrifuged at 14,000 rpm for 2 min. The resulting supernatant was centrifuged at 14,000 rpm for 3 min to pellet any cell debris. One volume of 70% ethanol was added to the cleared lysate, added to the RNeasy column and centrifuged at 10,000 rpm for 15 sec. Bound RNA was washed with the addition of RW1 buffer to the column and allowed to stand at room temperature for 10 min followed by centrifugation at 10,000 rpm for 15 sec. DNase 1 incubation mix (Stock: 273 kunits; RNase-free DNase Set, Qiagen) was added directly to the spin column membrane and digestion of any contaminating genomic DNA was carried out at room temperature for 30 min. The column was washed with RWI buffer and the DNase treatment step was repeated. The bound RNA was washed with RW1 buffer and centrifuged at 10,000 rpm for 15 sec three times, followed by two additional wash and centrifugation steps with RPE buffer. Finally, purified RNA was eluted off the column with RNase-free water. RNA concentration and purity was determined spectrophotometrically at A260/A280 and was determined to be intact as assessed by formaldehyde gel electrophoresis. The presence of genomic DNA contamination was assessed by PCR amplification of GAPDH using standard PCR conditions and the following primers: GAPDH_F2-5’- ACCCACTCCTCCACCTTTGAC-3’, GAPDH_R2- 5’-CTGTTGCTGTAGCCAAATTCGT-3’. RNA was re-treated with DNase if necessary. Upon confirming the quality of the RNA (intact and free of genomic DNA), 15 µg of total RNA from each of the five biological replicates was used for expression screening on either the Affymetrix or the clone-based filter platform. Keywords: parallel sample
2003-12-14 | GSE405 | GEO
Project description:MicroRNAs (miRNAs), small (18–22 nucleotides) non-coding RNAs, suppress the translation of target mRNAs by binding to their 3′ untranslated region. Evidence suggests that miRNAs are key regulators of several cellular processes, including angiogenesis. Recent findings indicate that Secreted miRNAs enclosed in exosomes also play an important role in cell–cell communication. To elucidate whether miRNAs secreted from neoplastic cells transfer into endothelial cells and are functionally active in the recipient cells, we investigated the interaction of a leukemia cell line and human umbilical vein endothelial cells (HUVECs). The culture medium was collected and centrifuged at 3000 × g for 15 min. The supernatant was filtered through a 0.22-µm PVDF filter (Millipore). The appropriate volume of Exoquick Exosome Precipitation Solution (System Biosciences, Mountain View, CA, USA) was added to the filtered culture medium and mixed well by inverting. After refrigeration for 12 h, the mixture was centrifuged at 1,500 × g for 30 min and all supernatant was removed by aspiration. Exosome pellets were resuspended with 500 µl of the serum-free AIM V medium (Invitrogen). K562 cells or K562/Cy3-miR-92a cells (K562 cells transfected with Cy3-labeled pre-miR-92a) were pre-cultured in serum-free AIM V medium (Invitrogen). The cells were cultured in 200 ml of AIM V medium (twenty 100 mm culture dishes). For exosome preparation, the culture media were collected and centrifuged at 2,000 × g for 10 min at room temperature, and the supernatant was centrifuged again at 12,000 × g for 30 min at room temperature, then the supernatant was ultracentrifuged at 110,000 × g for 70 min at 4 ºC. The pellets were washed with PBS, after ultracentrifugation, they were used for Microarray analysis.
2012-01-26 | E-GEOD-35256 | biostudies-arrayexpress
Project description:Cells were washed with cold PBS twice and scraped from the plates, then centrifuged at 1000 rpm for 5 min to pellet cells. Washed cells were lysed with 400 L of cell lysis buffer (10 mM Tris-HCl pH7.5, 150 mM NaCl, 0.15% NP-40 and proteinase inhibitor cocktail) and incubated on ice for 10 min. The suspension was carefully add at the top of sucrose buffer (10 mM Tris-HCl pH7.5, 150 mM NaCl, 24% sucrose), and centrifuged at 3200g for 10 min to collect nuclear pellets. Pellets were resuspended in 250 L of Glycerol buffer (20 mM Tris-HCl pH7.5, 75 mM NaCl, 0.5 mM EDTA pH8.0, 50% glycerol), then then immediately add 250 μl Nuclear lysis buffer (10 mM Tris-HCl pH7.5, 300 mM NaCl, 7.5 mM MgCl2, 0.2 mM EDTA pH8.0, 1% NP-40, 1M urea). Mix by vortexing for 4 s and incubate on ice for 2 min. Centrifuge the lysate at 13,000 × g for 2 min to precipitate the chromatin–RNA complex. Briefly rinse the chromatin pellets with PBS-EDTA. RNA was extracted with Trizol reagent. RNA libraries were prepared according to manual of SMARTer smRNA-Seq Kit for Illumina (Clontech, 635031).
2019-10-15 | GSE134103 | GEO
Project description:For the blood contamination studies a CSF pool was made with 1mL CSF free of blood from n=4 patients. The pool was divided into four aliquots. One aliquot was kept as reference CSF without added blood (named “neat” in the raw files), one was spiked with 20 µL blood/mL CSF (2%) (“20S”) and two were spiked with 5 µL blood/mL CSF (0.5%) (named “5S” and “5U”, S=centrifuged, U=not centrifuged). The sample spiked with 2% blood and one of the samples spiked with 0.5% blood were centrifuged at 4C at 400 x g for 10 minutes. In one experiment (BloodContamination_GeLC-MS_comb1-10) the reference CSF (neat), and 0.5% centrifuged (5S) and 2% centrifuged (20S) were protein depleted using the MARS Hu-14 column, separated by SDS-PAGE into ten fractions and in-gel digested. The samples (30 in total) were analysed by LC-MS on an OrbiTrap Velos Pro online coupled to a Dionex Ultimate 3000 nano RSLC system. The data was analysed by the Progenesis LC MS software 2.7 (Nonlinear Dynamics), and the MS/MS spectra were searched against UniProt/SwissProt using the open-source graphical user interface SearchGUI (version 1.7.3), with search engines OMSSA and X!Tandem. PeptideShaker (version 0.14.7) was used to assemble the peptides into proteins. The raw files were named according to sample and fraction, e.g. the first fraction of the reference CSF was called “BK_GeLC_neat_F1”, and the second fraction was called “BK_GeLC_neat_F2”. In the second blood contamination study the reference CSF (neat), and the 0.5% blood spiked samples centrifuged (5S) and not centrifuged (5U) were trypsin digested by in solution protocol and analysed using the same instruments as in the first study. (In the search output file are also the results for 2% blood spiked with and without centrifugation, 20S and 20U, but since the data was not used, the raw files are not distributed). The raw files were named “BK_Insol_FD_X” (X = neat, 5S or 5U). In the third experiment we examined the rostro-caudal gradient (RCG) on CSF in the spinal cord by sampling the 1st, 10th, 16th, 24th, 31st, 38th and 44th mL CSF in volumes of approximately 1 mL of a PSP patient during lumbar puncture. The CSF was centrifuged at 2000 x g for 10 min. We did an iTRAQ discovery study, and to be able to compare all seven RCG points, three related iTRAQ experiments (RCG exp 1, 2 and 3) were done. In each related experiment we included an identical reference which we labeled with the iTRAQ 114 reagent. The reference sample contained equal volumes of the seven RCG points, and was used as the reference in the data analysis. In the experiment we had twelve samples (equal volume) that were digested and labeled with iTRAQ reagents according to the vendor’s manual. The samples were combined into three related experiments as follows: Exp. 1 (common reference, 44th mL, 24th mL and 1st mL), Exp. 2 (common reference, 1st mL, 38th mL and 16th mL), and Exp. 3 (common reference, 10th mL, 44th mL and the 31st mL). The 1st and 44th mL were included twice, since they were expected to be the most different samples. The three combined samples (RCG exp 1, 2 and 3) were fractionated into 21 fractions using mixed mode reversed phase-anion chromatography (MM (RP-AX)). Fractions 1-4 were excluded from LC-MS analysis and the two latest fractions were combined before analysis on an Orbitrap Velos Pro, resulting in 16 fractions per combined sample. The raw files were named according to experiment (RCG 1, 2 or 3) and number of fraction (F4-F19), e.g. the raw file by the name EA_RCG3_F15 is fraction 15 from RCG experiment 3.
2014-02-13 | PXD000401 | Pride
Project description:Blood samples were collected from all participants in 10-mL EDTA-coated Vacutainer tubes. Plasma was separated by centrifugation at 3,000 rpm for 10 min at room temperature (25°C) within 2 h after blood collection and then centrifuged at 13,000 rpm for 10 min at 4°C to remove debris. Isolated plasma samples were stored at -80°C until use. EV RNA were isolated by affinity-based binding to spin columns using an exoRNeasy Serum/Plasma kit (Qiagen, Hilden, Germany). RNA libraries were prepared for sequencing using SMARTer® Stranded Total RNA-Seq Kit - Pico Input Mammalian. RNA-seq was performed by the Illumina sequencing platform on a 150-bp paired-end run.
2019-10-01 | GSE133684 | GEO
Project description:Preparation of AMDs. 125 μM Ac4ManNAz were incubated with Raw 264.7 (~ 107 cells/dish) for 24, 48, and 72 h, followed by gentle washing with PBS or complete medium to produce Raw 264.7-Ac4ManNAz. Subsequently, Raw 264.7-Ac4ManNAz (~ 107 cells) were resuspended in PBS containing Alkynyl-Ce6(0.1 μM), ascorbic acid (0.2 μM) and CuSO4 (0.2 μM) and incubated for 1h at 37 °C. Then centrifuged (1000 rpm, 3 min) and washed 3 times with PBS to obtain precursor of AMDs. precursor of AMDs (~ 107 cells/tube, 1.0 ml) was immersed in liquid nitrogen, 24 h later, the frozen tubes were thawed at 37°C, centrifuged at 1500 rpm for 3 min, washed and resuspended by PBS to generate the AMDs (5 x 106 units/tube, 1.0 ml).
2024-03-21 | PXD050063 | Pride
Project description:Samples were subjected to sonication using Bioruptor (program mode – 30 sec on, 30 sec off, 10 cycles = 10 min). Samples were sonicated till a clear solution was obtained. After a short spin (5,000 rpm for 10 sec), the samples were heated for 10 minutes at 95°C at 300 rpm in a PCR 96 heating block. Samples were allowed to cool down and spun at 5,000 rpm for 10 sec. 1μl of chloroacetamide was added to the eluate and incubated at 37°C for 30 minutes at 500 rpm. Later the samples were spun down at 5,000 rpm for 10 sec. Proteins were subjected to endoproteinase LysC (1:100 (w/w), Wako) digestion at 37°C for 4 hrs. Later, the proteins were subjected to trypsin digestion (0.5 μg/ μl; 1:50; w/w) at 37 °C overnight. Digestion was stopped by adding 50 μl of 5% TFA (Applied Biosystems) (v/v) that lowered the pH of the solution to below pH 2.0. Subsequently, peptides were cleaned up using the Phoenix 96x (https://preomics.com) kit following the manufacture’s instructions. After drying the peptides in a SpeedVac, samples were stored at -80°C.
2020-01-13 | PXD013159 | Pride
Project description:The goals of this study are to compare extracellular vesicles-derived circRNAs-lncRNAs-mRNA(RNA-seq) from the plasma of myocardial infarction patients with cardiac remodeling (C), without cardiac remodeling (NC), and normal healthy people (N). Human plasma was separated by centrifugation at 3,000 rpm for 10 min at room temperature (25°C) within 2 h after blood collection and then centrifuged at 13,000 rpm for 10 min at 4°C to remove debris. Isolated plasma samples were stored at -80°C until use. Extracellular vesicles RNA were isolated by affinity-based binding to spin columns using an exoRNeasy Serum/Plasma kit (Qiagen, Hilden, Germany). RNA libraries were prepared for sequencing using SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian. RNA-seq was performed by the Illumina NovaSeq 6000 on a 150-bp paired-end run.
2023-02-08 | GSE194388 | GEO
Project description:The chromatin fraction was isolated from 5x107 HeLa cells. Chromatin was digested in 100 mL of MNase (40 units/ mL ) reaction buffer for 3 min at 37 °C in a thermomixer (1,400 rpm). After addition of 10 L EGTA (25mM) to inactivate MNase, soluble digested chromatin was collected by 13,000 rpm centrifuge for 5 min. 10 mg of Pol II antibody was conjugated to magnetic protein A beads. The supernatant was diluted with 400 mL of NET-2 buffer and pSer2-Pol II antibody-conjugated beads were added. Incubated the IP reaction at 4 °C for 1 hr. The beads were washed with 1 mL of NET-2 buffer six times and 100 mL of 1xPNKT (1xPNK buffer and 0.05% Triton X-100) buffer once. Washed beads were incubated in 50 mL PNK reaction mix (1xPNKT, 1 mM ATP and 0.05 U/ml T4 PNK (NEB)) in Thermomixer at 37 °C, 1,400 rpm for 6 min. Beads were washed with 1 mL of NET-2 buffer once and RNA was extracted with Trizol reagent. RNA was suspended in urea Dye (7M Urea, 1xTBE, 0.1% BPB and 0.1% XC) and resolved on 6% TBU gel (Invitrogen) at 200 V for 5 min. Gels with 30-160 nt RNAs were collected. 0.2 mL tube was prepared with holes made with 25G needle and placed in a 1.5 mL tube. Gel fragments were placed in the layered tube and broken down by centrifugation at 12,000 rpm for 1 min. The small RNAs were eluted from gel using RNA elution buffer (1 M NaOAc and 1 mM EDTA) at 25 °C for 1 hr in Thermomixer (900 rpm). Eluted RNA was purified with SpinX column (Coster) with 2 glass filters (Millipore) and the flow-through RNA was ethanol precipitated.
2019-10-15 | GSE134102 | GEO
Project description:Using laser capture microdissection to isolate over 100 specific cell populations, we report the profiling of prostate cancer progression from benign epithelium to metastatic disease. By analyzing these expression signatures in the context of over 15,000 â??molecular conceptsâ??, or sets of biologically connected genes, we generated an integrative model of prostate cancer progression. Keywords: disease state analysis Experiment Design: â?¢ Type of experiment: This study used microarray experiments to profile prostate cancer progression through the isolation of pure cell populations using laser capture microdissection (LCM) and OmniPlex Whole Transcriptome (WTA) Amplification. â?¢ Experimental factors: This study profiled various pure cell populations isolated by LCM from prostate tissue. â?¢ The number of hybridizations performed in the experiment: A total of 104 hybridizations were performed. â?¢ The type of reference used for the hybridizations: A single commercially available pool of benign prostate tissue (CPP, Clontech) was used as the reference in all hybridizations. For the reference sample, 10 ng of CPP total RNA was converted into a WTA cDNA library in a volume of 25 µl and five 5 µl aliquots were amplified by WTA PCR and purified. Five ng aliquots of the pooled products were subjected to a second WTA PCR amplification in the presence of amino-allyl dUTP and all products were pooled before labeling to ensure equal representation across all of the hybridizations. â?¢ Hybridization design: For all hybridizations, the experimental prostate sample isolated by LCM was labeled with Cy5 and the common reference of benign pooled prostate as described above was labeled with Cy3. â?¢ Quality control steps taken: In order to assess biological and technical reproducibility, we captured identical regions from two specimens (PCA_11 and MET_HR_3) and subjected one sample, PCA_30, to two hybridizations. For PCA_11, adjacent regions of the same section were captured and treated as separate samples. For MET_HR_3, two separate foci of the liver metastasis (from separate frozen blocks) were captured and treated as separate samples. For PCA_30, two aliquots of the primary WTA PCR products were separately subjected to a second round of WTA PCR amplification. Replicates from PCA_30 and MET_HR_3 were also hybridized to arrays from each of the print runs utilized in this study. Samples used, extract preparation and labeling: â?¢ The origin of the biological sample (for instance, name of the organism, the provider of the sample) and its characteristics: All samples used were of human origin. The table in the MIAME Checklist (Supplmentary Methods) summarizes the samples used. Hybridizations are named according to the class of sample profiled and the background color is the same as shown in Figure S3. Two print runs were used during the course of the study and the indicated run for each hybridization is given. For prostate cancer samples, the Gleason patterns present on the sample section used are indicated, as well as the Gleason patterns of the actual cells isolated by LCM. A unique case number for all profiled samples is given. Descriptions of samples are given where applicable, including the location of metastatic foci. â?¢ Manipulation of biological samples and protocols used: Laser Capture Microdissection (LCM) was performed from frozen tissue sections with the SL Microtest device using µCUT software (MMI). Approximately 10,000 cells were captured for each sample. Serial sections were used if cells could not be obtained from a single section. Total RNA was isolated from captured cells with the RNAqueous Micro kit (Ambion) and treated with DNAse I according to the manufacturer's instructions. RNA quantification was perfomed using Ribogreen (Molecular Probes). â?¢ Protocol for preparing the hybridization extract: For each sample, total RNA (~10ng) in a volume of 25 µl was converted into an OmniPlex WTA cDNA library and amplified by WTA PCR using reagents and protocols according to the beta-commercial TransPlex WTA kit (Rubicon Genomics, Ann Arbor, MI). For each sample, a single 10 µl aliquot of the WTA cDNA library was amplified by WTA PCR and products were purified. Four or five 5 ng aliquots of product were subjected to a second WTA PCR amplification in the presence of amino-allyl dUTP for post amplification labeling and products were pooled before proceeding with the hybridization. Yields after all WTA PCR amplifications were between 2 to 5 μg per reaction, and reaction progress was monitored in real time using SYBR green I (Molecular Probes) on the iCycler IQ (BioRad) or ABI 7300 Real Time PCR system (Applied Biosystems). Real-time PCR amplifications were terminated at or before plateau phase as measured by fluorescence incorporation to preserve maximum representation. â?¢ Labeling protocol(s): For all WTA amplified cDNA samples with amino-allyl dUTP incorporated (10-20 ï?g), nuclease free water was added to 500 ï?l, the sample was transferred to a Microcon YM-30 filter, and centrifuged at 13,000 rcf at RT for 12 minutes. The spin column was inverted in a new collection tube and centrifuged at 13,000 rcf for 2 min at RT. The amount of sample was measured and the volume was adjusted to 5 ï?l with nuclease free water. For labeling, 5 ï?l of 1 M sodium bicarbonate (pH 9.0) was added and allowed to incubate at RT for 15 min. Nine ï?l of anhydrous DMSO were added to Mono Reactive Cy3 and Cy5 dye packs (Amersham, Buckinghamshire, England), mixed thoroughly, and 1 ï?l of the proper dye was added to each sample. The labeling mixture was incubated in the dark at RT for 1 hour. The reaction was stopped by the addition of 9 ï?l of 4 M Hydroxylamine for 15 min. For each labeling mixture, RNase free water was added to 100 ï?l, 500 ï?l of PB buffer was added and Cy3 and Cy5 mixtures were added to separate Qiaquick (Qiagen, Valencia, CA) spin columns and centrifuged at 13,000 rcf. Columns were washed twice with 700 ï?l of buffer PE, and centrifuged dry at 13,000 rcf for 2 min. To elute, 60 ï?l of EB buffer was placed on the column, incubated for 5 min and centrifuged at 13,000 rcf for 1 min. The Cy3 labeled and Cy5 labeled cDNA for each hybridization were mixed and 1.5 ï?l were used for analysis on a ND-1000 spectrophotometer. â?¢ External controls (spikes): Not utilized Hybridization procedures and parameters: â?¢ The protocol and conditions used during hybridization, blocking and washing: For hybridization, 40 ï?g of human Cot-1 DNA (Invitrogen, Carlsbad, CA) was added to 120 ï?l of labeled cDNA, the probe was transferred to a Microcon YM-30 filter and centrifuged at 13,000 rcf for 6 min at RT. The spin column was inverted in a new collection tube and centrifuged at 13,000 rcf for 2 min at RT. The eluted probe volume was adjusted to 18.6 ï?l with nuclease free water and the following were added: 4 ï?l of yeast tRNA (10 ï?g/ï?l, Invitrogen, Carlsbad, CA), 4.9 ï?l of 20X SSC and 0.84 ï?l of 10% SDS. The probe was denatured at 100 Co for 3 min and centrifuged at 13,000 rcf for 45 sec. The probe was added directly to the microarray and a cover slip was added. Slides were hybridized overnight in DIE-TECH 1 or 5 slide hybridization chambers in a 65 Co water bath. After hybridization, cover slips were removed by incubation in 2x SSC / 0.05% SDS. Slides were then washed in 2x SSC / 0.05% SDS for 5 min and 0.2x SSC / 0.05% SDS for five minutes. Slides were then washed in 0.2x SSC for 10 sec and centrifuged dry at 500 rpm for 5 min. Measurement data and specifications: â?¢ Type of scanning hardware and software used: Microarrays were scanned using a GenePix 4000B scanner (Axon Instruments, Union City, CA). â?¢ Type of image analysis software used: Images were gridded and spots were quantified using GenePix Pro 4.0 software (Axon Instruments, Union City, CA). â?¢ A description of the measurements produced by the image-analysis software and a description of which measurements were used in the analysis: The measurements made by GenePix Pro 4.0 are described in the sample files. For all hybridizations, the log2 normalized Median of Ratios (as described below) was used. â?¢ Data selection and transformation procedures: Arrays were auto-gridded by GenePix 4.0. Features flagged by GenePix as not found during grid
2010-06-05 | E-GEOD-6099 | biostudies-arrayexpress