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| Status |
Public on Feb 11, 2010 |
| Title |
Differential cytogenomics and miRNA signature of the Acute Myeloid Leukemia Kasumi-1 cell line CD34+38- compartment |
| Organism |
Homo sapiens |
| Experiment type |
Genome variation profiling by genome tiling array
Non-coding RNA profiling by genome tiling array
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| Summary |
The t(8;21) Acute Myeloid Leukaemia (AML) Kasumi-1cell line with N822K KIT mutation, is a model system for leukemogenesis. As AML initiating cells reside in the CD34+CD38– fraction, we addressed the refined cytogenomic characterization and miRNA expression of Kasumi-1 cell line and its FACS-sorted subpopulations focussing on this compartment. By conventional cytogenetics, Spectral Karyotyping and array-CGH the cytogenomic profile of Kasumi-1 cells evidenced only subtle regions differentially represented in CD34+CD38– cells.
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| Overall design |
Cell culture
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).
Immunophenotypic characterization
Kasumi-1 cells, after washing and blocking, were stained with the following antibodies: Cy7-phycoerythrine (PE) conjugated anti-CD34, PE conjugated anti-CD38, allophycocyanin (APC) conjugated anti-CD117, PE conjugated anti-CD33, fluorescein-5-isothiocyanate (FITC) anti -CD3, CD4, CD8, CD14, CD16, CD19, CD20 and FITC conjugated anti-CD13, CD235a, CD41 (Becton Dickinson, BD Biosciences). The analysis was performed using a FacsCalibur instrument (Becton Dickinson, BD Biosciences).
Cell sorting
Kasumi-1 cells were stained with anti-CD34 and anti-CD38 antibodies as described above. Cell sorting 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.
Nucleic acid extraction
DNA was isolated from FACS-sorted CD34-, CD34+CD38- Kasumi-1 subpopulations and Kasumi-1 total cell line 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.). DNA was conserved at -20°C until use.
Array CGH
An oligonucleotide array, containing 244,000 probes designed for human CGH, was utilized (Agilent Technologies, Palo Alto, CA). It is composed of 60-mer oligonucleotides at an average spatial resolution of 6.4 Kb.
Labelling and Hybridization of DNA: respectively 1μg of reference DNA (pooled normal female control) and test sample genomic DNA (either Kasumi-1 total cell line and sorted CD34- and CD34+CD38- cells) were individually fluorescently labelled using the Agilent Genomic DNA Labelling kit, Direct Method, as described by the manufacturer (Agilent Technologies, Palo Alto, CA). The labelled DNA was hybridized to the Agilent human 244K CGH array (Agilent Technologies, Palo Alto, CA) according to the manufacturer’s instructions. Chip was scanned with an Agilent microarray scanner to generate high-resolution (5 µm) images.
-Image and data Analysis: Image analysis was performed using the Feature Extraction version 9.5.1.1 (Agilent Technologies, Palo Alto, CA) and the Agilent’s CGH-v4_95_Feb07 protocol. The results of Feature Extraction were imported into CGH Analytics version 3.4.27 (Agilent Technologies, Palo Alto, CA). Aberration calls were made using Agilent’s ADM-1 algorithm with a threshold of 10, comparing the Kasumi-1 DNA sample versus the control DNA sample. Array CGH was performed by Empire Genomics, Buffalo, NY, USA.
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| Contributor(s) |
Pedranzini L, Mottadelli F, Ronzoni S, Rossella F, Ferracin M, Magnani I, Roversi G, Colapietro P, Negrini M, Pelicci PG, Larizza L |
| Citation(s) |
20227111 |
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| Submission date |
Feb 09, 2010 |
| Last update date |
Oct 26, 2016 |
| Contact name |
Jeffrey Conroy |
| E-mail(s) |
jeffrey.conroy@roswellpark.org
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| Organization name |
Roswell Park Cancer Institute
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| Street address |
Elm & Carlton Sts
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| City |
Buffalo |
| State/province |
NY |
| ZIP/Postal code |
14263 |
| Country |
USA |
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| Platforms (1) |
| GPL4091 |
Agilent-014693 Human Genome CGH Microarray 244A (Feature number version) |
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| Samples (3) |
| GSM506744 |
Acute Myeloid Leukemia Kasumi-1 cell line CGH |
| GSM506867 |
Acute Myeloid Leukemia Kasumi-1 CD34- cell line CGH |
| GSM506973 |
Acute Myeloid Leukemia Kasumi-1 CD34+CD38- cell line CGH |
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| Relations |
| BioProject |
PRJNA125741 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE20239_RAW.tar |
223.4 Mb |
(http)(custom) |
TAR (of TXT) |
| Processed data included within Sample table |
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