|
| Status |
Public on Jan 20, 2012 |
| Title |
3-GD2+ |
| Sample type |
RNA |
| |
|
| Source name |
human neuroblastoma cells
|
| Organism |
Homo sapiens |
| Characteristics |
disease status: neuroblastoma
patient stage: stage 4
cell type: Neuroblast freshly isolated GD2 positive cells, from bone marrow
|
| Treatment protocol |
fresh BM samples from patients with stage 4 NB older than 18 months at diagnosis were subjected to immunomagnetic separation, after lysis of erythrocytes, cells were incubated for 30 minutes at 4°C with the anti-GD2 mAb in the presence of human Fc blocking reagent. After washing, the cell pellet was incubated for 15 minutes at 4°C with goat anti-mouse IgG2a microbeads (Miltenyi). Cells were then washed twice, suspended in 500 uL of sterile ice-cold PBS and loaded onto a MS column (Miltenyi).The GD2 positive fractions were then subjected to RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated by using the RNAeasy Mini Kit (Qiagen) according to manufacturer’s protocol. Total RNA was quantified and quality control assessed by Nano LabChip® assay on the 2100 Bioanalyzer (Agilent Technologies). Only samples with a RNA Integrity Number >7 were included in the study.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 500 ng RNA as detailed in the One-color microarray-based gene expression analysis protocol, v5.5 (www.agilent.com). Dye incorporation and cRNA yield were checked with Nanodrop (Thermo Scientific).
|
| |
|
| Hybridization protocol |
Labelled cRNAs were hybridized to whole-genome oligonucleotide microarrays containing 41,000 human transcripts (Agilent Technologies) following the manufacture's instructions (One-color microarray-based gene expression analysis protocol, v5.5). Briefly, 1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer's instructions. On completion of the fragmentation reaction, 250 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome 4x44K Oligo Microarrays (G4112F) 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), 1 minute with 37°C GE Wash buffer 2 (Agilent), with the Stabilization solution (Agilent) for 1 minute at room temperature, then dried immediately by rinsing slides into Drying solution (Agilent) for 30 seconds.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to XDR Hi 100% and XDR Lo 10%). Microarray performance was assessed by QC metric tool.
|
| Description |
Gene expression
|
| Data processing |
The scanned TIFF images were analyzed with Feature Extraction Software version 9.5.3.1 (Agilent) using default parameters (Agilent protocol GE1-v5_95_Feb07) to obtain background subtracted and spatially detrended Processed Signal intensities. The output of Feature Extraction was analyzed with the Agi4x44PreProcess package. After within-array median-summarization of probe intensities within replicates, probe intensities were log base 2 transformed and normalized across arrays with the quantile normalization method implemented in Bioconductor.
Bioconductor normalized data are provided as supplementary files on the Series record.
|
| |
|
| Submission date |
Nov 26, 2010 |
| Last update date |
Jan 20, 2012 |
| Contact name |
PAOLA SCARUFFI |
| E-mail(s) |
paola.scaruffi@hsanmartino.it
|
| Organization name |
"San Martino" Hospital
|
| Lab |
Center of Physiopathology of Human Reproduction
|
| Street address |
LARGO R. BENZI, 10
|
| City |
Genova |
| State/province |
GE |
| ZIP/Postal code |
16132 |
| Country |
Italy |
| |
|
| Platform ID |
GPL4133 |
| Series (1) |
| GSE25623 |
Bone marrows infiltrating human neuroblastoma cells express high levels of calprotectin and HLA-G proteins. |
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