|
| Status |
Public on Jul 03, 2015 |
| Title |
ITD2_1 |
| Sample type |
RNA |
| |
|
| Source name |
FLT3-ITD AML patient sample
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: FLT3-ITD AML patient sample
|
| Treatment protocol |
none
|
| Growth protocol |
Patient and donor samples were obtained either from blood or bone marrow. Lymphoprep extraction of leukocytes was performed following 30 min no brake centrifugation. Enrichment for CD34 and CD117 were checked via FACS analysis. For CD14 samples, enricment for CD14 was controlled by FACS analysis. Where available, purification was performed using MACS columns following anti-CD34 bead conjugation to samples (anti-CD14 for CD14 samles).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from cells using TRIzol® (Invitrogen) according to manufacturers instruction. First strand cDNA synthesis was carried out using M-MLV reverse transcriptase (Invitrogen) according to manufacturers instructions using 1-2 μg of RNA. Real Time PCR was carried out using ABI SYBR® green master mix with 2 μl of diluted cDNA and 0.25 μM primer per 10 μl reaction on an ABI 7500 machine.
|
| Label |
Cy3
|
| Label protocol |
25 ng RNA was labelled with Cyanine 3-CTP according to the sample preparation protocol from Agilent: One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling). Amplified labelled RNA samples were purified by using Qiagen's RNeasy mini spin columns and cRNA quantified by using a Nanodrop
|
| |
|
| Hybridization protocol |
Hybridisation samples were prepared for a 8-pack microarray using 600 ng cRNA each according to the hybridisation protocol from Agilent: One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling), loaded onto the 8-pack array slide and hybridised at 65 °C overnight
|
| Scan protocol |
After washing the microarray was scanned on an Agilent C Scanner using the Profile AgilentG3_GX_1Color for 8x60K microarrays (Dye channel: Green; Scan region: Scan Area (61 x 21.6 mm); Scan resolution (μm): 3; Tiff: 20 bit)
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.1.1 (Agilent) (protocol GE1_107_Sep09, Grid: 028004_D_F_20110325 and platform Agilent SurePrint G3 Human GE 8x60K). The raw data output by Feature Extraction Software was analysed using the LIMMA R package with quantile normalisation and background subtraction.
|
| |
|
| Submission date |
Jan 12, 2015 |
| Last update date |
Jul 04, 2015 |
| Contact name |
Pierre Daniel Cauchy |
| E-mail(s) |
cauchy@ie-freiburg.mpg.de
|
| Phone |
+49 (0)761 270-77576
|
| Organization name |
University Medical Center Freiburg
|
| Department |
Zentrum für Translationale Zellforschung
|
| Lab |
AG Onco-Immunology
|
| Street address |
Breisacher Str. 115
|
| City |
Freiburg |
| State/province |
Baden-Württemberg |
| ZIP/Postal code |
79106 |
| Country |
Germany |
| |
|
| Platform ID |
GPL13607 |
| Series (2) |
| GSE64873 |
Chronic growth factor signaling in Acute Myeloid Leukemia is connected to a specific chromatin signature [Agilent] |
| GSE64874 |
Chronic growth factor signaling in Acute Myeloid Leukemia is connected to a specific chromatin signature |
|
