|
| Status |
Public on Nov 22, 2016 |
| Title |
HSC_ITD_Adult_16471 |
| Sample type |
RNA |
| |
|
| Source name |
Bone marrow
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
developmental stage: Adult
genotype: FLT3 ITD/+
tissue: HSC
|
| Treatment protocol |
No treatments. Cells were harvested directly from mice by flow cytometry
|
| Growth protocol |
HSCs and HPCs harvested directly from mouse by flow cytometry
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using the Qiagen RNAeasy micro plus columns
|
| Label |
Cy5
|
| Label protocol |
RNA samples were amplified using SIGMA WTA2 RNA amplification system. Amplified cDNAs were chemically labeled with Kreatech ULS RNA labeling kit (Kreatech Diagnostics). Per reaction, 3ug of each RNA (+water=16ul) was mixed with Kreatech 10x labeling buffer (2ul) and Kreatech cy5/DY-ULS (2ul). The reactions were incubated at 85C for 15 minutes in the dark and placed on ice for 3 minutes. Labeled cDNA was purified with Qiagen PCR purification columns according to manufacturer’s protocol. cDNAs were quantitated on a Nanodrop spectrophotometer. Detailed protocol can be found at http://www.kreatech.com/fileadmin/user_upload/Documenten/PDF/12_man_EA-021-022-023__D0.6.pdf
|
| |
|
| Hybridization protocol |
The balanced aRNAs were suspended in Agilent 2X Gene Expression buffer (55ul), Agilent 10X Blocking agent (11ul), and Kreablock (6ul). The hybridization solutions were applied to Agilent Mouse v2 4x44K microarrays (G4846A-026655). Hybridization was carried out at 65C for 20 hours. Washing procedures were carried out according to Agilent gene expression protocols.
|
| Scan protocol |
Slides were scanned on an Agilent C-class Microarray scanner to detect Cy5 fluorescence, according to manufacturer's specifications.
|
| Description |
Gene expression in HSCs
|
| Data processing |
Gridding and analysis of images was performed using Feature Extraction (v11.5.1.1, Agilent Technologies).
|
| |
|
| Submission date |
May 05, 2016 |
| Last update date |
Nov 22, 2016 |
| Contact name |
Jeffery Magee |
| E-mail(s) |
magee_j@kids.wustl.edu
|
| Organization name |
Washington University School of Medicine
|
| Department |
Internal Medicine - Cardiovascular Division
|
| Street address |
660 S Euclid Ave.
|
| City |
St Louis |
| State/province |
Missouri |
| ZIP/Postal code |
63110 |
| Country |
USA |
| |
|
| Platform ID |
GPL10787 |
| Series (1) |
| GSE81153 |
Fetal and neonatal hematopoietic progenitors are functionally and transcriptionally resistant to Flt3-ITD mutations. |
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