|
| Status |
Public on Dec 31, 2011 |
| Title |
CLL FG_co-culture_48h_rep8 |
| Sample type |
RNA |
| |
|
| Source name |
CLL co-cultured 48h on HUVEC layer 48hr
|
| Organism |
Homo sapiens |
| Characteristics |
tissue origin: peripheral blood
cell type: Chronic lymphocytic leukemic B cells (CLL)
|
| Growth protocol |
Purified CD19+ CLL cells were suspended at a final concentration of 1 x 106/ml in AIM-V medium (Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum and then plated in 24-well plates (CLL only). For co-culture experiments, HUVEC cells were incubated until reaching 70% confluent and CLL cells were then seeded onto HUVEC layer at a final concentration of 1 x 106/ml in AIM V supplemented with 10% FBS (CLL HC). At the indicated time points, CLL cells were collected by removal of the supernatant leaving HUVEC cells intact and then being assayed for cell viability and gene expression profiling.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
total RNAs were extracted from purified CD19+ cells using RNeasy Midi kit Plus (QIAGEN, Valencia, CA, USA) following the manufacturer's recommendations. The protocol includes an on-column DNase digestion. RNAs were quantified using InfiniteM200 (Tecan).
|
| Label |
Cy3
|
| Label protocol |
High quality RNAs were amplified and Cy3-labeled using Low Input Quick Amp Labeling kit (Agilent Technologies, Palo alto, CA, USA). Agilent RNA One-Color Spike-In was added in each sample to provide positive controls for monitoring the microarray workflow from sample amplification and labelling to microarray processing. It contains 10 in vitro synthesized, polyadenylated transcripts derived from the Adenovirus E1A transcriptome that are premixed at various ratios. All cRNA products were purified using RNeasy columns (QIAGEN). Dye incorporation and cRNA yield were checked with InfiniteM200 (Tecan).Samples had to contain at least 6 pmole of cyanine dye/ug of cRNA to be considered suitable for subsequent hybridization.
|
| |
|
| Hybridization protocol |
Cy3-labeled cRNAs (1.65 ug) were fragmented to an average size of 50-100 nt by incubation at 60°C for 30 min using in situ Hybridization kit-plus (Agilent). Samples were hybridized for 17 hours at 65°C on 4x44K Whole Human Genome Microarray (Agilent).
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner 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 100%).
|
| Description |
Gene expression of CLL cells collected from peripheral blood and then cultured on endothelial cell layer for 48 hours
|
| Data processing |
Fluorescence data were analyzed with Feature Extraction Software v.10.5 (Agilent Technologies) an QC Chart tool v.1.3 to obtain background subtracted, spatially detrended Processed Signal intensities and quality control analyses. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Apr 21, 2011 |
| Last update date |
Dec 31, 2011 |
| Contact name |
Rossana Maffei |
| E-mail(s) |
rossana.maffei@unimore.it
|
| Phone |
+39 059 4222715
|
| Organization name |
University of Modena and Reggio Emilia
|
| Department |
Dept of Hematology and Oncology
|
| Lab |
Lab of Molecular Hematology
|
| Street address |
Via del Pozzo 71
|
| City |
Modena |
| State/province |
Modena |
| ZIP/Postal code |
41100 |
| Country |
Italy |
| |
|
| Platform ID |
GPL6480 |
| Series (1) |
| GSE28787 |
Interaction between endothelium and chronic lymphocytic leukemia B-cells rescues from apoptosis and modulates gene expression profile of leukemic cells |
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