|
| Status |
Public on Nov 16, 2008 |
| Title |
GBM Cell B vs Exosome B |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Primary glioblastoma cells
|
| Organism |
Homo sapiens |
| Characteristics |
Primary human glioblastoma cells cultured in vitro.
|
| Growth protocol |
Glioblastoma cells at passage 1–15 were grown for 48 hrs in DMEM with exosome-depleted 5% FBS. After 48 hrs, the supernatant was collected for exosome purification, and in parallel, the cells were harvested for RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The cells were washed in PBS and then subjected to total RNA extraction with the MirVana RNA isolation kit according to manufacturer's recommendation.
|
| Label |
Cy3
|
| Label protocol |
For the linear T7-based amplification step, 1 μg of each total RNA and 0.5 μg of each exosome RNA sample was used as starting material. To produce Cy3- and Cy5-labeled cRNA, the RNA samples were amplified and labeled using the Agilent Low RNA Input Linear Amp Kit (Agilent Technologies) following the manufacturer’s protocol.
|
| |
|
| Channel 2 |
| Source name |
Exosomes shed from primary human glioblastoma cells
|
| Organism |
Homo sapiens |
| Characteristics |
Exosomes were isolated from the glioblastoma cells analyzed in channel 1.
|
| Growth protocol |
Glioblastoma cells at passage 1–15 were cultured in microvesicle-free medium (DMEM containing 5% dFBS) and conditioned medium was collected after 48 h. Microvesicles were purified by differential centrifugation. In brief, glioblastoma-conditioned medium was centrifuged for 10 min at 300g to eliminate cell contamination. Supernatants were further centrifuged for 20 min at 16,500g and filtered through a 0.22 μm filter. Microvesicles were pelleted by ultracentrifugation at 110,000g for 70 min. The microvesicle pellets were washed in 13 ml PBS, pelleted again and resuspended in PBS before RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The exosomes were washed in PBS and then subjected to total RNA extraction with the MirVana RNA isolation kit according to manufacturer's recommendation.
|
| Label |
Cy5
|
| Label protocol |
For the linear T7-based amplification step, 1 μg of each total RNA and 0.5 μg of each exosome RNA sample was used as starting material. To produce Cy3- and Cy5-labeled cRNA, the RNA samples were amplified and labeled using the Agilent Low RNA Input Linear Amp Kit (Agilent Technologies) following the manufacturer’s protocol.
|
| |
|
| |
| Hybridization protocol |
The Agilent array data was performed by Miltenyi Biotech.
The hybridization procedure was performed according to the Agilent 60-mer oligo microarray processing protocol using the Agilent Gene Expression Hybridization Kit (Agilent Technologies). Briefly, 825 ng of the corresponding Cy3- and Cy5-labeled fragmented cRNA were combined and hybridized overnight (17 hours, 65 °C) to Agilent Whole Human Genome Oligo Microarrays 4x44K using Agilent’s recommended hybridization chamber and oven. Finally, the microarrays were washed once with 6x SSPE buffer containing 0.005% Nlauroylsarcosine for 1 min at room temperature followed by a second wash with pre-heated 0.06x SSPE buffer (37 °C) containing 0.005% N-lauroylsarcosine for 1 min. The last washing step was performed with acetonitrile for 30 sec.
|
| Scan protocol |
Fluorescence signals of the hybridized Agilent Oligo Microarrays were detected using Agilent’s DNA microarray scanner (Agilent Technologies).
|
| Description |
The Agilent Feature Extraction Software (FES) determines feature intensities and ratios (including background subtraction and normalization), rejects outliers and calculates statistical confidences (p-values). The Rosetta Resolverâ software offers – among other features – the possibility to visualize the results of the data analysis in a double-log scatter plot.
|
| Data processing |
The Agilent FES was used to read out and process the microarray image files. The VALUE columns are based on the Agilent ProcessedSignal values that have gone through the Agilent Feature Extraction steps. For determination of differential gene expression, FES-derived output data files were further analyzed using the Rosetta Resolver gene expression data analysis system (Rosetta Biosoftware). Data was analyzed using the GeneSifter software.
|
| |
|
| Submission date |
Nov 04, 2008 |
| Last update date |
Nov 06, 2008 |
| Contact name |
Johan Skog |
| Organization name |
Massachusetts General Hospital
|
| Department |
Molecular Neurogenetics
|
| Lab |
XOB
|
| Street address |
149 13th street
|
| City |
Charlestown |
| State/province |
MA |
| ZIP/Postal code |
02129 |
| Country |
USA |
| |
|
| Platform ID |
GPL4133 |
| Series (1) |
| GSE13470 |
Glioblastoma microvesicles transport RNA and proteins |
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