|
| Status |
Public on Oct 06, 2014 |
| Title |
Primary_human_uveal_melanocyte_cells_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
Primary human uveal melanocyte cells, replicate 3
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Primary human uveal melanocyte cells
gender: male
age: 83 years
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using the RNeasy Mini Kit (QIAGEN, Toronto, ON) from HCEC, HSEC, HCFC, HSFC, UVM isolated from normal eyes supplied by the National Eyes Bank from the CHU de Québec (QC, Canada), and from the uveal melanoma cell lines T115, T142 and T143 cultured from the primary tumors (115, 142 and 143) of patients diagnosed with uveal melanoma .
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 50 ng RNA using the One-Color LowInput QuickAmp Labeling Kit One-Color (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Toronto, ON). Dye incorporation and cRNA yield were checked with the Nanophotometer pearl(Montréal Biotech).
|
| |
|
| Hybridization protocol |
600 ng of Cy3-labelled cRNA (specific activity >8.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole SurePrint G3 Human GE 8x60K (G4851A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent SureScanner (G4900DA) using one color scan setting for 1x60k array slides (Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10,7 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Oct 06, 2014 |
| Last update date |
Oct 06, 2014 |
| Contact name |
Karine Zaniolo |
| E-mail(s) |
karine.zaniolo@gmail.com
|
| Organization name |
Centre de recherche du CHU de Québec
|
| Department |
CUO-Recherche
|
| Lab |
Sylvain Guérin
|
| Street address |
1050 Chemin Sainte-Foy
|
| City |
Québec |
| State/province |
Québec |
| ZIP/Postal code |
G1S 4L8 |
| Country |
Canada |
| |
|
| Platform ID |
GPL13607 |
| Series (1) |
| GSE62075 |
Characterization of the human a9 integrin subunit gene: promoter analysis and transcriptional regulation |
|
