|
| Status |
Public on Oct 31, 2010 |
| Title |
Lipid A treated (4h) - biological replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Bone marrow derived macrophages (BMDM)
|
| Organism |
Mus musculus |
| Characteristics |
Bone marrow derived macrophages (BMDM)
age: 6 weeks
strain: C56/Bl6 mice
|
| Treatment protocol |
Treated in vitro with 100ng/ml Lipid A(LPA) for 4h
|
| Growth protocol |
Non-adherant bone-marrow cells (2x106 cells) were plated into 6-well plates(Nuncleon, Denmark) in 2ml of DMEM complemented with 10% FCS and 20% (v/v) L929-conditioned medium as a source of MCSF and placed in the incubator at 37C, 5% CO2 humified atmosphere. 1ml of medium was replaced from each well and replenished with fresh medium supplemented with 20% L929-conditioned medium every 2 days. At day 7, the cells formed an adherent monolayer which resembled macrophages in their morphology and showed 98% purity by F4/80 staining in FACS. At this stage, cells were washed with complete medium and ready for treatment.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions, miRNA extracted using microcon YM-100 fractionation followed by isopropanol precipitation.
|
| Label |
Cy3
|
| Label protocol |
500ng of miRNA were labeled using LABELIT® kit (Mirus, USA) according to manufacturer's protocol.
|
| |
|
| Channel 2 |
| Source name |
Bone marrow derived macrophages (BMDM)-Dcr1 hypomorphic
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/5; Dcr1 hypomorphic mice
age: 6 weeks
|
| Treatment protocol |
Treated in vitro with 100ng/ml Lipid A(LPA) for 4h
|
| Growth protocol |
Non-adherant bone-marrow cells (2x106 cells) were plated into 6-well plates(Nuncleon, Denmark) in 2ml of DMEM complemented with 10% FCS and 20% (v/v) L929-conditioned medium as a source of MCSF and placed in the incubator at 37C, 5% CO2 humified atmosphere. 1ml of medium was replaced from each well and replenished with fresh medium supplemented with 20% L929-conditioned medium every 2 days. At day 7, the cells formed an adherent monolayer which resembled macrophages in their morphology and showed 98% purity by F4/80 staining in FACS. At this stage, cells were washed with complete medium and ready for treatment.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions, miRNA extracted using microcon YM-100 fractionation followed by isopropanol precipitation.
|
| Label |
Cy5
|
| Label protocol |
500ng of miRNA were labeled using LABELIT® kit (Mirus, USA) according to manufacturer's protocol.
|
| |
|
| |
| Hybridization protocol |
Labeled miRNA targets and hybridization buffer were added, and samples were applied to microarrays and hybridized at 42C for 16 hrs, using Telechem hybridization chambers.
|
| Scan protocol |
Scanned on an Axon 4000B and analysed using Axon Genepix 4.0
|
| Description |
Biological replicate 2 of 2. DICER knockout, treated, harvested after 4hr treatment.
|
| Data processing |
LOWESS normalized, background subtracted VALUE data obtained from log2 of processed Red signal/processed Green signal. Acuity software was used.
|
| |
|
| Submission date |
Sep 08, 2008 |
| Last update date |
Dec 03, 2009 |
| Contact name |
Peter Nissom |
| E-mail(s) |
pnissom@gmail.com
|
| Organization name |
Bioprocessing Technology Institute
|
| Street address |
20 Biopolis Way
|
| City |
Singapore |
| ZIP/Postal code |
138668 |
| Country |
Singapore |
| |
|
| Platform ID |
GPL7257 |
| Series (1) |
| GSE12699 |
Murine bone-marrow derived macrophages: Wild type Control vs. DICER1 (Dcr1) hypomorphic cells. |
|
