|
| Status |
Public on Nov 13, 2008 |
| Title |
Non-adherent cells pool vs. Adherent cells pool |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Non-adherent cells pool
|
| Organism |
Homo sapiens |
| Characteristics |
Non-adherent cells after one HSCs co-culture on MSCs feeder layer
|
| Growth protocol |
Hematopoietic stem cells (HSCs) were suspended in HSC serum-free medium supplemented with 150 ng/ml Fetal Liver Tyrosine Kinase-3 ligand (FLT3-L), 150 ng/ml stem cell factor (SCF) and 50 ng/ml IL-3 for one week
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using standard RNA extraction protocols (NucleoSpin® RNA II, Macherey-Nagel)
|
| Label |
Cy3
|
| Label protocol |
the RNA samples were amplified and labeled using the Agilent Low RNA Input Linear Amp Kit (Agilent Technologies) following the manufacturer’s protocol. Yields of cRNA and the dye-incorporation rate were measured with the ND-1000 Spectrophotometer (NanoDrop Technologies).
|
| |
|
| Channel 2 |
| Source name |
Adherent cells pool
|
| Organism |
Homo sapiens |
| Characteristics |
Adherent cells after one HSCs co-culture on MSCs feeder layer
|
| Growth protocol |
Hematopoietic stem cells (HSCs) were suspended in HSC serum-free medium supplemented with 150 ng/ml Fetal Liver Tyrosine Kinase-3 ligand (FLT3-L), 150 ng/ml stem cell factor (SCF) and 50 ng/ml IL-3 for one week
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using standard RNA extraction protocols (NucleoSpin® RNA II, Macherey-Nagel)
|
| Label |
Cy5
|
| Label protocol |
the RNA samples were amplified and labeled using the Agilent Low RNA Input Linear Amp Kit (Agilent Technologies) following the manufacturer’s protocol. Yields of cRNA and the dye-incorporation rate were measured with the ND-1000 Spectrophotometer (NanoDrop Technologies).
|
| |
|
| |
| Hybridization protocol |
The hybridization procedure was performed according to the Agilent 60-mer oligo microarray processing protocol using the Agilent Gene Expression Hybridization Kit (Agilent Technologies). Briefly, 825 ng of the corresponding Cy3- and Cy5-labeled fragmented cRNA were combined and hybridized overnight (17 hours, 65 °C) to Agilent Whole Human Genome Oligo Microarrays 4x44K using Agilent’s recommended hybridization chamber and oven. Finally, the microarrays were washed once with 6x SSPE buffer containing 0.005% Nlauroylsarcosine for 1 min at room temperature followed by a second wash with pre-heated 0.06x SSPE buffer (37 °C) containing 0.005% N-lauroylsarcosine for 1 min. The last washing step was performed with acetonitrile for 30 sec.
|
| Scan protocol |
Fluorescence signals of the hybridized Agilent Oligo Microarrays were detected using Agilent’s DNA microarray scanner (Agilent Technologies)
|
| Description |
All samples were shipped to Miltenyi Biotec (Bergisch Gladbach, Germany) which performed RNA extraction, RNA quality control, Hybridization of Agilent Whole Genome Oligo Microarrays, Fluorescence signals scanning and data analysis.
|
| Data processing |
The Agilent Feature Extraction Software (FES) was used to read out and process the microarray image files. The software determines feature intensities and ratios (including background subtraction and normalization), rejects outliers and calculates statistical confidences (p-values). For determination of differential gene expression FES derived output data files were further analyzed using the Rosetta Resolver® gene expression data analysis system (Rosetta Biosoftware).
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| |
|
| Submission date |
Nov 12, 2008 |
| Last update date |
Nov 13, 2008 |
| Contact name |
Nael Alakel |
| E-mail(s) |
[email protected]
|
| Organization name |
university hospital Dresden
|
| Street address |
Fetscherstrasse 74
|
| City |
Dresden |
| ZIP/Postal code |
01307 |
| Country |
Germany |
| |
|
| Platform ID |
GPL4133 |
| Series (1) |
| GSE13566 |
Direct contact with mesenchymal stromal cells impacts CD133+ hematopoietic stem cells during ex-vivo expansion |
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