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| Status |
Public on Apr 16, 2010 |
| Title |
SBC-3_exosome_rep2 |
| Sample type |
RNA |
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| Source name |
SBC-3, exosome, replicate 2
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| Organism |
Homo sapiens |
| Characteristics |
cell line type: small cell lung carcinoma
fraction: exosome
cell line: SBC-3
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| Biomaterial provider |
JCRB
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| Treatment protocol |
Cells were cultured in complete RPMI-1640 medium (Sigma) for 24 h at 37°C and 5% of CO2, washed twice with phosphate-buffered saline (PBS), and incubated 48 h in phenol red-free RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum which was previously depleted contaminating microvesicles by overnight centrifugation at 100,000×g. Culture medium was collected and centrifuged at 800×g for 5 min and additional 2,000×g for 10 min to remove lifted cells. For isolation of exosomes, the supernatant was subjected to filtration on a 0.1 μm pore polyethersulfone membrane filter (Corning, Corning, NY) to remove cell debris and large vesicles, followed by concentration by CentriPlus-70 with a 100,000 Mw cut-off membrane (Millipore, Billerica, MA). The supernatant was then ultracentrifuged at 100,000×g for 1 h at 4°C using 70Ti rotor (Beckman Coulter, Brea, CA). The resulting pellets were resuspended in PBS and ultracentrifuged at 100,000×g for 1 h at 4°C using 100Ti rotor (Beckman Coulter). For RNA isolation from culture medium, after cells were grown for 48 h as described above, culture medium was collected and centrifuged at 800×g for 5 min and additional 2,000×g for 10 min. The supernatant was subjected to filtration on a 0.22 μm pore polyethersulfone membrane filter (Corning), followed by concentration by CentriPlus-70 with a 5,000 Mw cut-off membrane (Millipore). The supernatant was mixed with equal volume of QIAzol Lysis Reagent (Qiagen) and followed the procedure for RNA isolation described below. For RNA isolation from cells, cells were harvested by treatment of Trypsin-EDTA (Invitrogen) and washed twice with PBS (pH 7.4).
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| Growth protocol |
Cancer cell lines including AZ-521, AZ-P7a, NIH-H69, SBC-3, DMS53, SW480, and SW620 were grown in RPMI1640 medium (Sigma, St. Louis, MO) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen, Carlsbad, CA), 100 U/ml penicillin, and 0.1 mg/ml of streptomycin in a humidified 5% CO2 incubator at 37°C.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells, exosomes, and supernatant fractions from culture medium were subjected to isolation of total RNA using miRNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocols. The protocols include digestion of genomic DNA with RQ1 RNase-Free DNase (Promega) in the presence of RNasin Plus RNase Inhibitor (Promega, Madison, WI) and the following purification with the RNeasy MinElute Cleanup Kit (Qiagen). RNA was quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Wilmington, DE) and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
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| Label |
Cy3
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| Label protocol |
Cyanine-3 (Cy3) labeled miRNA was prepared from 100 ng for cellular RNA analysis and 20 ng for extracellular RNA analysis using the miRNA Complete Labeling Reagent and Hyb Kit (Agilent) according to the manufacturer's instructions (Agilent microRNA microarrays version 2.2).
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| Hybridization protocol |
Cy3-labelled miRNA was hybridized to Agilent Human miRNA Microarray Kit (8×15K) Ver3.0 (G4470C) for 20 hours at 55°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 5 minute at room temperature with GE Wash Buffer 1 (Agilent) and 5 minute at 37°C with GE Wash buffer 2 (Agilent), then dried.
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| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565BA) using one color scan setting for 8x15k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to 100%, No XDR).
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| Description |
miRNA expression in exosome of SBC-3
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| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol miRNA_107_Sep09 and Grid: 021827_D_F_20091031) to obtain background subtracted and spatially detrended Processed Signal intensities.
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| Submission date |
Apr 15, 2010 |
| Last update date |
Nov 15, 2012 |
| Contact name |
Keiichi HATAKEYAMA |
| E-mail(s) |
[email protected]
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| Organization name |
Shizuoka Cancer Center Research Institute
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| Street address |
1007 Shimonagakubo
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| City |
Nagaizumi-cho |
| State/province |
Sunto-gun, Shizuoka |
| ZIP/Postal code |
411-8777 |
| Country |
Japan |
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| Platform ID |
GPL14767 |
| Series (1) |
| GSE21350 |
miRNA signatures of intracellular and extracellular fractions from cancer cell lines |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM533533_252182711131_201002161343_S01_miRNA_107_Sep09_1_4.txt.gz |
826.2 Kb |
(ftp)(http) |
TXT |
| Processed data provided as supplementary file |
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