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Sample GSM622848 Query DataSets for GSM622848
Status Public on Apr 16, 2012
Title HMC-1_exosome_RNA_4
Sample type RNA
 
Source name exosomes isolated from the supernatant of HMC-1 cells
Organism Homo sapiens
Characteristics cell line: mast cell line HMC-1
total rna source: exosome
Treatment protocol Growth under normal conditions
Growth protocol Cell culture and exosome release The human mast cell line HMC-1 (Dr Joseph Butterfield, Mayo Clinic, USA), was cultured in IMDM containing 10% depleted fetal bovine serum (FBS), 100 U ml-1 penicillin, 100 µg ml-1 streptomycin, 2 mM L-glutamine and 1.2 mM alpha-thioglycerol (all from Sigma-Aldrich). To eliminate exosomes present in serum, FBS was ultracentrifuged at 120 000 g for 90 min using a Ti70 rotor (Beckman optima LE-80k Ultracentrifuge). Peripheral blood mononuclear cells (PBMC) were prepared from peripheral blood of healthy subjects by Ficoll-Plaque density separation. The CD34 cells were obtained from the PBMC by positive isolation using magnetic separation according to the manufacturer’s instruction (Miltenyi Biotec, Germany). CD34 cells were cultured in IMDM (Sigma-Aldrich) containing 10% FBS, 100 U ml-1 penicillin, 100 µg ml-1 streptomycin, 2 mM L-glutamine and 1mM sodium pyruvate. The purity of the separations ranged between 60-85%, as analyzed by flow cytometry using antibody against CD34 (BD Biosciences) in combination with 7-AAD for viability. Exosomes were isolated according to Valadi et al., 2007. Briefly, cells were stimulated with 1 µM calcium ionophore (Sigma-Aldrich) for 30 min, centrifuged at 500 g for 10 min to eliminate cells and at 16 500 g for 20 min, followed by filtration through 0.22 µm filter to remove cell debris. Exosomes were pelleted by ultracentrifugation (Beckman Ti70 rotor) at 120 000 g for 70 min. For PKH67 transfer experiments, exosomes were washed once in a large volume PBS. For electron microscopy, the exosome pellet was diluted in a large volume PBS, filtrated through 0.1 µm filters and pelleted by ultracentrifugation. Exosomes were measured for their protein content using BCA™ Protein Assay Kit (Pierce). Cell viability was assessed using trypan blue exclusion or 7-AAD.
Extracted molecule total RNA
Extraction protocol Extraction of total RNA was performed using Trizol according to the manufacturer's instructions.
Label biotin
Label protocol Biotinylated cRNA were prepared according to the standard Affymetrix protocol (Expression Analysis Technical Manual, 2001, Affymetrix). The experiment was performed by SweGene (http://www.lth.se/index.php?id=7789) according to the Affymetrix guidelines.
 
Hybridization protocol The experiment was performed by SweGene (http://www.lth.se/index.php?id=7789) according to Affymetrix guidelines. Briefly, a hybridisation cocktail was prepared with the biotinylated and fragmented cRNA, control oligonucleotide, herring sperm DNA, acetylated BSA, 4-morpholinepropanesulfonic acid (MES), Tween 20, and bacterial cRNA controls respectively. The hybridisation cocktail was heated to 99°C for 5 min and to 45°C for 5 min, and was briefly centrifuged before hybridization. The pre-wet GeneChipTM probe array cartridge was filled with the hybridisation cocktail and incubated on rotation, 60 rpm at 45°C for 16–18 h. The cartridge was then subjected to an automated washing procedure, using the GeneChipTM Fluidics Station 400 and with a non stringent wash buffer, containing saline, sodium, phosphate, EDTA (SSPE), Tween 20 and antifoam according to the Affymetrix manual. A final wash with the stringent buffer, containing MES-sodium salt and Tween 20 was performed. The probe array was then stained with a solution of acetylated BSA, streptavidin and R-phycoerythrin in stain buffer, containing sodium-MES and Tween 20 and antifoam, followed by non stringent wash buffer. A second stain was performed with a solution of acetylated BSA, normal goat IgG and biotinylated goat antistreptavidin antibody in stain buffer. A third staining step with streptavidin R-phycoerythrin was performed as described above, before a final one with non-stringent wash buffer at 30°C.
Scan protocol The probe arrays were scanned with the Gene Array Scanner (Affymetrix, Inc. www.affymetrix.com) and controlled by the software GenChip Operating System 1.2 (GCOS; Affymtrix Inc.).
Description p0739_E4.CEL
Data processing The expression level Signals were scaled in GCOS 1.2 to give a median array intensity of 100. This was done to enable different arrays to be compared. The program Spotfire DecisionSite 8.2 (www.spotfire.com) was used for gene-profiling analysis, genes only present in exosomes and expression differences. For the network analysis to identify biological mechanisms, the program Ingenuity was used (www.ingenuity.com).
 
Submission date Nov 12, 2010
Last update date Apr 16, 2012
Contact name Karin Ekström
E-mail(s) karin.ekstrom@biomaterials.gu.se
Organization name Krefting Research Centre
Street address Medicinaregatan 1G
City Gothenburg
ZIP/Postal code 40530
Country Sweden
 
Platform ID GPL570
Series (1)
GSE25320 Characterisation of mRNA and microRNA in human mast cell exosomes and their transfer to other mast cells and blood CD34 progenitor cells

Data table header descriptions
ID_REF
VALUE Signal
ABS_CALL indicating whether the transcript was present (P), absent (A), or marginal (M)
DETECTION P-VALUE

Data table
ID_REF VALUE ABS_CALL DETECTION P-VALUE
AFFX-BioB-5_at 3181.16 P 5.16732e-05
AFFX-BioB-M_at 6372.84 P 4.42873e-05
AFFX-BioB-3_at 3518.28 P 4.42873e-05
AFFX-BioC-5_at 12403 P 4.42873e-05
AFFX-BioC-3_at 14529.1 P 4.42873e-05
AFFX-BioDn-5_at 27289.8 P 4.42873e-05
AFFX-BioDn-3_at 36541.5 P 4.42873e-05
AFFX-CreX-5_at 113242 P 5.16732e-05
AFFX-CreX-3_at 128123 P 4.42873e-05
AFFX-DapX-5_at 3248.39 P 5.16732e-05
AFFX-DapX-M_at 11109.4 P 5.16732e-05
AFFX-DapX-3_at 23181.6 P 4.42873e-05
AFFX-LysX-5_at 559.583 P 7.00668e-05
AFFX-LysX-M_at 1527.72 P 4.42873e-05
AFFX-LysX-3_at 4665.95 P 4.42873e-05
AFFX-PheX-5_at 816.208 P 4.42873e-05
AFFX-PheX-M_at 2007.05 P 4.42873e-05
AFFX-PheX-3_at 2335.41 P 4.42873e-05
AFFX-ThrX-5_at 313.963 P 0.00179591
AFFX-ThrX-M_at 1808.39 P 4.42873e-05

Total number of rows: 54675

Table truncated, full table size 1587 Kbytes.




Supplementary file Size Download File type/resource
GSM622848.CEL.gz 3.9 Mb (ftp)(http) CEL
GSM622848.CHP.gz 300.7 Kb (ftp)(http) CHP
Processed data included within Sample table
Processed data provided as supplementary file

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