|
| Status |
Public on Apr 29, 2015 |
| Title |
HEL_NCD38_48h_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
HEL, NCD38, 48h, replicate3
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEL
cell type: leukemia
|
| Treatment protocol |
NCD38 or NCD25 was dissolved in Dimethyl Sulfoxide (DMSO). 5-7x10^5 Cells were seeded in 6 well plates. 2 micromolar NCD38, NCD25 or DMSO was added and incubated for 48 hours at 37 ºC in the incubator with 5% CO2.
|
| Growth protocol |
HEL and CMK11-5 were cultured in 10% heat-inactivated fetal bovine serum (FBS) containing RPMI medium. MDS-L was cultured in the same medium with 20ng/ml human IL-3 (PEPRO TECH) and 20M 2-mercaptoethanol (Gibco)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using RNA easy mini kit (Qiagen) following the manufacturer's recommendations. RNA was quantified using the NanoDrop-1000 spectrophotometer. Quality of total RNA was evaluated with the Agilent 2100 Bioanalyzer (Agilent Technologies).
|
| Label |
Cy3
|
| Label protocol |
150ng total RNA was labeled with Cyanine-3 (Cy3) using Low Input Quick Amp Labeling Kit, one-color (Agilent) according to the manufacturer’s protocol, followed by RNAeasy column purification (QIAGEN). Dye incorporation was checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
600ng Cy3-Labeled cRNA was fragmented using Gene Expression Hybridization Kit (Agilent) according to the manufacturer’s protocol and hybridized to SurePrint G3 Human GE Microarray 8 x 60K Ver2.0 (G4858A) (Agilent) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed with GE Wash Buffer 1 and Buffer 2 (Agilent).
|
| Scan protocol |
Microarray slides were scanned immediately after washing on the Microarray Scanner G2565BA (Agilent) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression of HEL after 48hr incubation with NCD38
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1_1100_Jul11 and Grid: 039494_D_F_20120628) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Apr 28, 2015 |
| Last update date |
Apr 29, 2015 |
| Contact name |
Masahiro Kawahara |
| E-mail(s) |
machakm6@kuhp.kyoto-u.ac.jp
|
| Phone |
+81757514964
|
| Organization name |
Kyoto University
|
| Department |
Hematology Oncology
|
| Lab |
leukemia research
|
| Street address |
54 Shogoin-Kawahara-cho, Sakyo-ku,
|
| City |
Kyoto |
| State/province |
State... |
| ZIP/Postal code |
6068507 |
| Country |
Japan |
| |
|
| Platform ID |
GPL17077 |
| Series (1) |
| GSE68348 |
Differential gene expression profiling by LSD1 inhibition using novel LSD1 inhibitors (NCD25 and NCD38) |
|
