|
| Status |
Public on Aug 16, 2011 |
| Title |
R765_LAF_CD34+ |
| Sample type |
RNA |
| |
|
| Source name |
CD34+ cells from Leucapheresis
|
| Organism |
Homo sapiens |
| Characteristics |
gender: female
|
| Treatment protocol |
digestion with collagenase and purification with CD34+ beads for Lipotransfer aspirates; mobilization of bone marrow cells with growth factors and purification with CD34+ cells for Leucapheresis
|
| Growth protocol |
none
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extraction with QiaAmp RNA blood mini kit with DnaseI Rnase-free treatment.
|
| Label |
biotin
|
| Label protocol |
Biotin-labelled cDNA targets were synthesized starting from 200 ng of total RNA. Double stranded cDNA synthesis and related cRNA was performed with GeneChip WT cDNA Synthesis and Amplification Kit . With the same kit was synthesized the sense strand cDNA before to be fragmented and labelled with GeneChip WT Teminal Labeling Kit.. All steps of the labelling protocol were performed as suggested by Affymetrix (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).
|
| |
|
| Hybridization protocol |
Hybridization mix for target dilution (100 mM MES, 1 M [Na+], 20 mM EDTA, 0.01% Tween 20) was prepared as indicated by Affymetrix, including DMSO at a final concentration of 7% and pre-mixed biotin-labelled control oligo B2 and bioB, bioC, bioD and cre controls (Affymetrix cat #900299) at a final concentration of 50 pM, 1.5 pM, 5 pM, 25 pM and 100 pM, respectively. Targets were diluted in hybridization buffer at a concentration of 25 ng/ul and denatured at 99 °C for 5 minutes and centrifuged at maximum speed for 1 minute prior to introduction into the GeneChip cartridge. A single GeneChip Human Gene 1.0 ST was then hybridized with each biotin-labelled sense target. Hybridizations were performed for 16 h at 45 °C in a rotisserie oven. GeneChip cartridges were washed and stained with GeneChip Hybridization, Wash and Stain Kit in the Affymetrix fluidics station following the FS450_0007 standard protocol, including the following steps: (1) (wash) 10 cycles of 2 mixes/cycle with Wash Buffer A at 30 °C; (2) (wash) 6 cycles of 15 mixes/cycle with Wash Buffer B at 50 °C; (3) stain of the probe array for 5 min in SAPE solution at 35 °C; (4) (wash) 10 cycles of 4 mixes/cycle with Wash Buffer A at 30 °C; (5) stain of the probe array for 5 min in antibody solution at 35 °C; (6) stain of the probe array for 5 min in SAPE solution at 35 °C; (7) (final wash) 15 cycles of 4 mixes/cycle with Wash Buffer A at 35 °C; (8) fill the probe array with Array Holding buffer.
|
| Scan protocol |
Images were scanned using an Affymetrix GeneChip Scanner3000 7G, using default parameters. The resulting images were analysed using GeneChip Operating Software v1.2 (GCOS1.2).
|
| Description |
R765_LAF_CD34+_(HuGene-1_0-st-v1).CEL
|
| Data processing |
The data were preprocessed with RMA using Partek GS software from Partek Inc., St.Louis, Missouri, USA using default settings. All analysis were performed considering only 28829 probesets belonging to the category main
probe group file: HuGene-1_0-st-v1.r4.pgf
meta-probeset file: HuGene-1_0-st-v1.na30.1.hg19.probeset.csv
|
| |
|
| Submission date |
Aug 16, 2011 |
| Last update date |
Aug 16, 2011 |
| Contact name |
Giuliana Gregato |
| E-mail(s) |
[email protected]
|
| Phone |
+390257489535
|
| Fax |
+390294379236
|
| URL |
http://www.ieo.it
|
| Organization name |
IEO
|
| Department |
Anatomy pathology
|
| Lab |
Oncohematology
|
| Street address |
Via ripamonti 435
|
| City |
Milan |
| ZIP/Postal code |
20141 |
| Country |
Italy |
| |
|
| Platform ID |
GPL6244 |
| Series (1) |
| GSE31415 |
Comparison of WAT CD34+ vs LAF CD34+ |
|
