|
| Status |
Public on Mar 22, 2011 |
| Title |
HMC1 17h SerumStarv 4h Imatinib versus HMC1 17h SerumStarv 4h Imatinib 2h NGF |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
HMC1 17h SerumStarv 4h Imatinib
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HMC-1
|
| Treatment protocol |
HMC-1(V560G c-Kit) cells were serum starved for 17 h, then treated with dimethyl sulfoxide (DMSO) or 5 μM Imatinib for 4 hours prior to stimulation with 100 ng/ml mouse recombinant NGF. After 30 or 120 min the stimulation was stopped in ice-cold PBS. Samples from cells grown in medium supplemented with 10% FCS were analyzed in parallel.
|
| Growth protocol |
HMC-1(V560G c-Kit) cells were grown in RPMI1640 medium supplemented with 10% FCS.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Residual DNA contamination was removed with DNAseI (Invitrogen GmbH, Darmstadt, Germany) according to the manufacturer’s recommendations, and the RNA was again purified with RNeasy Mini kit (Qiagen).
|
| Label |
Cy3
|
| Label protocol |
1000ng of total RNA per condition were used to prepare Cy3-, or Cy5-labeled cRNA with the “Low RNA Input Linear Amplification Kit PLUS, Two-Color” (#5188-5340, Agilent Technologies) according to the manufacturer’s recommendations.
|
| |
|
| Channel 2 |
| Source name |
HMC1 17h SerumStarv 4h Imatinib 2h NGF
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HMC-1
|
| Treatment protocol |
HMC-1(V560G c-Kit) cells were serum starved for 17 h, then treated with dimethyl sulfoxide (DMSO) or 5 μM Imatinib for 4 hours prior to stimulation with 100 ng/ml mouse recombinant NGF. After 30 or 120 min the stimulation was stopped in ice-cold PBS. Samples from cells grown in medium supplemented with 10% FCS were analyzed in parallel.
|
| Growth protocol |
HMC-1(V560G c-Kit) cells were grown in RPMI1640 medium supplemented with 10% FCS.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Residual DNA contamination was removed with DNAseI (Invitrogen GmbH, Darmstadt, Germany) according to the manufacturer’s recommendations, and the RNA was again purified with RNeasy Mini kit (Qiagen).
|
| Label |
Cy5
|
| Label protocol |
1000ng of total RNA per condition were used to prepare Cy3-, or Cy5-labeled cRNA with the “Low RNA Input Linear Amplification Kit PLUS, Two-Color” (#5188-5340, Agilent Technologies) according to the manufacturer’s recommendations.
|
| |
|
| |
| Hybridization protocol |
cRNA fragmentation, hybridization and washing steps were performed exactly as recommended by the manufacturer “Two-Color Microarray-Based Gene Expression Analysis Protocol V5.5” except that 4 µg of each labeled cRNA were used for hybridization.
|
| Scan protocol |
Slides were scanned on the Agilent Micro Array Scanner G2505 B at two different PMT settings (100 % and 5 %) to increase the dynamic range of the measurements (extended dynamic range mode).
|
| Data processing |
Data extraction and normalization were performed with the “Feature Extraction Software V9.5.3.1” by using the recommended default extraction protocol file: GE2-v5_95_Feb07.xml.
|
| |
|
| Submission date |
Mar 18, 2011 |
| Last update date |
Mar 22, 2011 |
| Contact name |
Oliver Dittrich-Breiholz |
| E-mail(s) |
dittrich.oliver@mh-hannover.de
|
| Organization name |
Medical School Hannover
|
| Department |
Research Core Unit Genomics
|
| Street address |
Carl-Neuberg-Str. 1
|
| City |
Hannover |
| ZIP/Postal code |
30625 |
| Country |
Germany |
| |
|
| Platform ID |
GPL4133 |
| Series (1) |
| GSE28045 |
Identification of novel target genes of nerve growth factor (NGF) in human mastocytoma cell line (HMC-1 (V560G c-Kit)) by transcriptome analysis |
|
