|
| Status |
Public on Nov 19, 2011 |
| Title |
MSC from patient B240 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
MSC from pediatric patient B240
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: mesenchymal stem cell (MSC)
disease state: pediatric severe aplastic anemia (SAA)
patient identifier: B240
|
| Treatment protocol |
Patients who had appropriate donors were having a bone transplantation; otherwise, they received anti-thymocyte immunoglobulins.
|
| Growth protocol |
Mesenchymal stem cells (MSCs) were kept in an undifferentiated, pluripotent state by growing in DMEM with low glucose concentration.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol following the manufacturer's instructions.
|
| Label |
Cy5
|
| Label protocol |
cRNA was synthesized from 0.5 μg of total RNA, and the RNA was amplified (Fluorescent Linear Amplification Kit, Agilent Technologies, Santa Clara, CA) and labeled with Cy3-CTP or Cy5-CTP (PerkinElmer, Waltham, MA). The cRNA of MSC from SAA patients was labeled with Cy5 and the cRNA from the control group was labeled with Cy3. The Cy-labeled cRNA was fragmented to about 50-100 nucleotides long by incubation with fragmentation buffer (Agilent Technologies, Santa Clara, CA) at 60°C for 30 minutes.
|
| |
|
| Channel 2 |
| Source name |
Pooled MSC from healthy donors
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: mesenchymal stem cell (MSC)
disease state: healthy
sample type: reference
|
| Treatment protocol |
Patients who had appropriate donors were having a bone transplantation; otherwise, they received anti-thymocyte immunoglobulins.
|
| Growth protocol |
Mesenchymal stem cells (MSCs) were kept in an undifferentiated, pluripotent state by growing in DMEM with low glucose concentration.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol following the manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
cRNA was synthesized from 0.5 μg of total RNA, and the RNA was amplified (Fluorescent Linear Amplification Kit, Agilent Technologies, Santa Clara, CA) and labeled with Cy3-CTP or Cy5-CTP (PerkinElmer, Waltham, MA). The cRNA of MSC from SAA patients was labeled with Cy5 and the cRNA from the control group was labeled with Cy3. The Cy-labeled cRNA was fragmented to about 50-100 nucleotides long by incubation with fragmentation buffer (Agilent Technologies, Santa Clara, CA) at 60°C for 30 minutes.
|
| |
|
| |
| Hybridization protocol |
The fragmented Cy-labeled cRNA was pooled, and oligoarray control targets and hybridization buffer (Agilent In Situ Hybridization Kit Plus) were added. The samples were applied to Agilent Whole Human Genome 4x44k oligo microarrays (Agilent Technologies, Santa Clara, CA) enclosed in Agilent SureHyb-enabled hybridization chambers and hybridized at 60°C for 17 h. After hybridization, slides were washed sequentially to remove non-specifically binding residues.
|
| Scan protocol |
The hybridized microarrays were scanned with an Agilent microarray scanner (Agilent Technologies, Santa Clara, CA) at OD535 for Cy3 and OD625 for Cy5.
|
| Description |
SAA B240
MSC from pediatric SAA patient vs. pooled MSC from healthy donors.
|
| Data processing |
Scanned images were analyzed by Feature extraction software 8.1 (Agilent Technologies, Santa Clara, CA). After image analysis, the data were normalized by a rank-consistency-filtering LOWESS method, which was included in the package software of Feature extraction software 10.5.1.1 (Agilent Technologies, Santa Clara, CA).
|
| |
|
| Submission date |
Nov 18, 2011 |
| Last update date |
Nov 21, 2011 |
| Contact name |
Kuan-Chih Chow |
| E-mail(s) |
kcchow@dragon.nchu.edu.tw
|
| Phone |
+886-4-22840896-118
|
| Fax |
+886-4-22859270
|
| Organization name |
National Chung Hsing University
|
| Department |
Graduate Institute of Biomedical Sciences
|
| Lab |
Cancer Research Lab.
|
| Street address |
250 Kuo Kuang Road
|
| City |
Taichung |
| ZIP/Postal code |
40227 |
| Country |
Taiwan |
| |
|
| Platform ID |
GPL4133 |
| Series (1) |
| GSE33812 |
Gene expression profiles of bone marrow mesenchymal stem cells in pediatric patients with severe aplastic anemia |
|
