|
| Status |
Public on Mar 09, 2022 |
| Title |
CB 1 HSC (K16/2886) |
| Sample type |
RNA |
| |
|
| Source name |
cord blood
|
| Organism |
Homo sapiens |
| Characteristics |
flt3 mutational status: FLT3 WT
phenotype: CD45dim/CD34+/CD38-
gender: Not applicable
core-binding factor leukemia: Negative
wt1 overexpression: Negative
cell type: HSC
|
| Treatment protocol |
Mononuclear cells collected from AML patients and healthy controls were used to sort CD34+CD38+ and CD34+CD38- populations. If the number of CD34-positive cells concerned less than 50% of the total white blood cell population, CD34-isolation was performed using the CD34 MicroBead Kit (Milteny). The immature myeloid compartment was defined by CD34, CD45 and scatter properties. CD34+CD38+ blasts and CD34+CD38- stem cells were gated using lymphocytes and fluorescence-minus-one controls to determine CD38 expression cut-offs. Lymphocytes were sorted based on high CD45 expression and low SSC-A. Delineated cell populations were backgated on SSC-A/FSC-A and SSC-A/CD45 scatter plots to exclude non-specific events. Sorted cells were collected in RPMI supplemented with 50% fetal calf serum and a post-sort purity of >90% was reached. Following, cells were spun down (10 min, 3000 rpm, 4° C) and resuspended in TRIzol for RNA and/or DNA extraction.
|
| Growth protocol |
At diagnosis, mononuclear cells were isolated from bone marrow or peripheral blood by Ficoll density gradient (Axis-shield) and cryopreserved in 90% fetal calf serum and 10% dimethylsulfoxide. Samples were thawed, followed by 30 min incubation at room temperature (RT) in 20 mL RPMI with 20% FCS, 200 µL DNase I (1 mg/mL, grade II bovine pancreas) and 200 µL MgCl2 (1 M) (Sigma-Aldrich). After incubation, cells were spinoculated (10 min, 400 rpm) and washed once more with RPMI/20% fetal calf serum.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the miRNeasy Mini Kit (Qiagen) in combination with on-column DNase I digestion (RNase-Free DNase set, Qiagen) according to manufacturer’s instructions.
|
| Label |
Cy3
|
| Label protocol |
Amplified RNA (Complete Whole Transcriptome Amplification Kit, Sigma-Aldrich) was labelled using the Genomic DNA ULS Labeling Kit (Agilent).
|
| |
|
| Hybridization protocol |
Labeled RNA was hybridized to the array in combination with CGH blocking to reduce background signaling.
|
| Scan protocol |
Micro-arrays were analyzed using an Agilent micro-array scanner and Feature Extraction software (v12.0).
|
| Data processing |
Probe intensities were background subtracted, quantile-normalized and log2-based probe intensities were calculated. A target was present if the log2 expression value exceeded the cut-off set at 6.75, based on the dark corner control probe value plus 1.
|
| |
|
| Submission date |
Mar 11, 2019 |
| Last update date |
Mar 09, 2022 |
| Contact name |
Barbara Depreter |
| E-mail(s) |
barbara.depreter@ugent.be
|
| Organization name |
University of Ghent
|
| Department |
Internal Medicine and Pediatrics
|
| Street address |
C. Heymanslaan 10
|
| City |
Gent |
| State/province |
Not in US |
| ZIP/Postal code |
9000 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL18641 |
| Series (1) |
| GSE128103 |
Differential gene expression in leukemic blasts and leukemic stem cells (LSC) sorted from pediatric AML patients compared to control myeloblasts and hematopoietic stem cells (HSC) sorted from cord blood |
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