|
| Status |
Public on Feb 11, 2010 |
| Title |
Acute Myeloid Leukemia Kasumi-1 CD34+CD38- cell line CGH |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Kasumi-1, CD34+CD38-
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: Kasumi-1
gender: male
facs-sorted: CD34+CD38- Kasumi-1 subpopulation
|
| Biomaterial provider |
Laura Pedranzini
|
| Treatment protocol |
Kasumi-1 cells, after washing and blocking, were stained with Cy7-phycoerythrine (PE) conjugated anti-CD34 antibody and PE conjugated anti-CD38, (Becton Dickinson, BD Biosciences). Cell sorting of CD34+CD38- subpopulation was performed using a Facs Vantage SE Laser 488nm Interprise Coherent flow cytometer (Becton Dickinson, BD Biosciences), and gates were set up to exclude not viable cells and debris. The purity of sorted fractions was assessed to ensure sort quality.
|
| Growth protocol |
The human AML Kasumi-1 cell line (AML M2, AML1-ETO) was obtained from DMSZ (Braunschweig GERMANY) and maintained in RPMI 1640 (PBI International, Milan, Italy) with 10% fetal bovine serum (FBS; HyClone Laboratories, Logan, UT, USA).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was isolated from Kasumi-1 CD34+CD38- subpopulation cells using the NucleoSpin Tissue kit (Macherey-Nagel, Gmbh & Co. KG) according to the manufacturer's instructions. DNA quality and concentration were evaluated using spectrophotometer NANODROP 1000 ( Thermo Fischer Scientific Inc.).
|
| Label |
Cy5
|
| Label protocol |
Agilent Genomic DNA Labelling kit, Direct Method, as described by the manufacturer (Agilent Technologies, Palo Alto, CA). 1ug template
|
| |
|
| Channel 2 |
| Source name |
reference control
|
| Organism |
Homo sapiens |
| Characteristics |
gender: female
phenotype: normal pooled
tissue: peripheral blood
|
| Biomaterial provider |
Roswell Park Cancer Institute
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Qiagen FlexiGene DNA kit protocol (Cat# 51206).
|
| Label |
Cy3
|
| Label protocol |
Agilent Genomic DNA Labelling kit, Direct Method, as described by the manufacturer (Agilent Technologies, Palo Alto, CA). 1ug template
|
| |
|
| |
| Hybridization protocol |
1ug of reference and test sample genomic DNA were individually fluorescently labeled using the Agilent Genomic DNA Labeling kit, Direct Method, as described by the manufacturer (Agilent Technologies). Initially, the DNA is denatured in the presence of the random primer at 95C for 3 minutes in a thermalcycler, and then quickly cooled to 4C. The tubes are transferred to ice and labeling occurs with the addition of dUTP-cyanine 3 mix (or dUTP-cyanine 5) and Exo-Klenow fragment. Incubation takes place for two hours at 37C in a thermalcycler. After purification, the labeled DNA is combined and resuspended in 50ug Cot-1 DNA, and 1x Agilent blocking agent and hybridization buffer. The entire sample is heated to 95C for 3 minutes followed by a 37C incubation for 30 minutes. Hybridization to the Agilent human 244K CGH array was performed for 40 hours at 65C using a rotating hybridization oven. Arrays were washed according to the manufacturer's recommendations; air dried, and scanned using an Agilent DNA microarray scanner (Agilent Technologies, Inc), and processed using Agilent's Feature Extraction software.
|
| Scan protocol |
Arrays were scanned using an Agilent DNA microarray scanner (Agilent Technologies, Inc) to generate high-resolution (5 um) images for both Cy3 (control) and Cy5 (test) channels.
|
| Description |
Image analysis was performed using the Feature Extraction version 9.5.1.1 (Agilent Technologies) and the Agilent's CGH-v4_95_Feb07 protocol.
|
| Data processing |
Data was processed from Agilent's Feature Extraction output by CGH Analytics version 3.4.27 (Agilent Technologies). Aberration calls were made using Agilent's ADM-1 algorithm with a threshold of 10, comparing the Kasumi-1 CD34+CD38- DNA sample versus the control DNA sample.
|
| |
|
| Submission date |
Feb 05, 2010 |
| Last update date |
Feb 10, 2010 |
| Contact name |
Jeffrey Conroy |
| E-mail(s) |
jeffrey.conroy@roswellpark.org
|
| Organization name |
Roswell Park Cancer Institute
|
| Street address |
Elm & Carlton Sts
|
| City |
Buffalo |
| State/province |
NY |
| ZIP/Postal code |
14263 |
| Country |
USA |
| |
|
| Platform ID |
GPL4091 |
| Series (1) |
| GSE20239 |
Differential cytogenomics and miRNA signature of the Acute Myeloid Leukemia Kasumi-1 cell line CD34+38- compartment |
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