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| Status |
Public on Dec 11, 2016 |
| Title |
ExVivoExpanded GF+SDF1 stimulated HSPC_S4_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
ExVivoExpanded GF+SDF1-stimulated HSPC
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Ex Vivo expanded HSPC
gender: female
donor: Healthy Donor
|
| Treatment protocol |
Nucleated cells were isolated from mobilized peripheral blood stem cell harvests obtained during hematopoietic stem cell transplantation (Allogenic transplantation, n=4; Autologous transplantation, n=1). CD34+ hematopoietic stem and progenitor cells (HSPC) were enriched from PBSC harvests by immunomagnetic separation using negative selection method. Enriched CD34+ cells at 2.5 x 104/ ml were cultured in IMDM containing 10%FCS in a 96-well plate culture plate. The cultures were treated with recombinant TPO (50 ng/ml, Stem Cell Technology, Canada), SCF (50ng/ml), Flt3L (50 ng/ml) and SDF1 peptide (10ng/ml; A gift from Prof Nobukata Fujii and Dr Shinya Oishi, Kyoto University, Japan) for 4 days. Medium alongwith factors was replenished after 4 days. After 8 days of stimulation, cells were counted for total nucleated cell count and stored in RNAlater for microarray experiments .
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using Qiagen’s RNeasy minikit as per the manufacturer’s protocol
|
| Label |
Cy3
|
| Label protocol |
The samples for Gene expression were labeled using Agilent Quick-Amp labeling Kit (p/n5190-0442). Five hundred nanograms each of the Control and test samples were incubated with reverse trancription mix at 40°C and converted to double stranded cDNA primed by oligodT with a T7 polymerase promoter. Synthesized double stranded cDNA were used as template for cRNA generation. cRNA was generated by in vitro transcription and the dye Cy3 CTP(Agilent) was incorporated during this step. The cDNA synthesis and in vitro transcription steps were carried out at 40°C. Labeled cRNA was cleaned up and quality assessed for yields and specific activity.
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| |
|
| Hybridization protocol |
The labeled cRNA samples were hybridized on to a Genotypic designed Custom Whole Genome Human 8x60k (AMADID No: 027114). 600ng of cy3 labeled samples were fragmented and hybridized. Fragmentation of labeled cRNA and hybridization were done using the Gene Expression Hybridization kit of Agilent (Part Number 5188-5242). Hybridization was carried out in Agilent’s Surehyb Chambers at 65º C for 16 hours. The hybridized slides were washed using Agilent Gene Expression wash buffers (Part No: 5188-5327)
|
| Scan protocol |
Slides were scanned using the Agilent Microarray Scanner G2505C at 3 micron resolution.
|
| Description |
Gene expression after ex-vivo expansion after stimulation of CD34-enriched HSPC with GF (SCF+TPO+FLT3) + homing
|
| Data processing |
Data extraction from Images was done using Feature Extraction software Version 10.5 and 75th Percentile shift Normalization of the data was done in GeneSpring GX V 11.0
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| |
|
| Submission date |
Apr 17, 2012 |
| Last update date |
Dec 11, 2016 |
| Contact name |
Jyoti Anand Kode |
| E-mail(s) |
[email protected]
|
| Phone |
+91-22-68735030
|
| Organization name |
Advanced Centre for Treatment, Research & Education in Cancer (ACTREC), Tata Memorial Centre
|
| Department |
Kode Lab
|
| Lab |
Tumor Immunology & Immunotherapy Group
|
| Street address |
Kharghar
|
| City |
Navi Mumbai |
| State/province |
Maharashtra |
| ZIP/Postal code |
410210 |
| Country |
India |
| |
|
| Platform ID |
GPL13252 |
| Series (1) |
| GSE37334 |
Gene Expression Profiling of Ex-vivo expanded Hematopoietic Stem and Progenitor Cells using SCF+TPO+FLT3L and homing factor SDF1 |
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