|
| Status |
Public on Aug 01, 2009 |
| Title |
Traf1_IFN+_B21B TF_RNAi-EXON_180407 |
| Sample type |
RNA |
| |
|
| Source name |
BMDMs, IFNg stimulation, siRNA treatment
|
| Organism |
Mus musculus |
| Characteristics |
Balb/c male mice age 10-12 weeks. Bone marrow (containing primary mouse monocytes) was flushed from femurs and differentiated for 7 days in DMEM-F12/10% FCS/10% L929 medium (releasing CSF-1). Cells grown in 24-well plates at a seeding density of 5x105 cells/well. Media changes on days 3 and 5.
|
| Treatment protocol |
Transfection of siRNA and IFNγ treatment Differentiated primary macrophages were subject to siRNAs SMARTpools (Thermo Fisher Inc) designed to each mRNA target (4 siRNAs per pool). siRNAs were purchased at a 5 nmol scale and redissolved in 1x siRNA buffer (Thermo Fisher Inc) to a final concentration of 1 µM. To transfect at a final concentration of 20 nM, 1 µl of siRNA SMARTpool was used with 49 µl of Optimem (Invitrogen, CA, USA) solution while 2 µl of Lipofectamine 2000 (L2K, Invitrogen) was mixed with 48 µl Optimem. Following incubation for 5 min, the siRNA mix was added to the L2K mix and incubated for a further 30 min, after which 400 µl of DMEM-F12/10% FCS/L929 medium lacking antibiotics was added to the siRNA:L2K complexes. Growth medium was removed and cells were washed 1x in PBS before 500 µl of the siRNA:L2K liposomes were added. Cells were then incubated for a further 24 h (37ºC, 5% CO2). For IFNγ treatments, growth medium was replaced with medium containing 10 U/ml recombinant mouse IFNγ. Cells were incubated for 24 h prior to harvesting of total RNA.
|
| Growth protocol |
Balb/c male mice age 10-12 weeks. Bone marrow (containing primary mouse monocytes) was flushed from femurs and differentiated for 7 days in DMEM-F12/10% FCS/10% L929 medium (releasing CSF-1). Cells grown in 24-well plates at a seeding density of 5x105 cells/well. Media changes on days 3 and 5.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using an RNeasy Plus kit (Qiagen) according to manufacturer's instructions. RNA was quantified and quality controlled using a NanoDrop spectrophotometer (NanoDrop Technologies) and BioAnalyser 2100 (Agilent). They were then washed and scanned according to manufacturer's recommendations
|
| Label |
biotin
|
| Label protocol |
Microarray target labelling: 150 ng of total RNA was processed using the Exon array target labelling kit (Affymetrix, Santa Clara, US) according to manufacturer’s instructions for small sample labelling without the use of the RiboMinus step.
|
| |
|
| Hybridization protocol |
Hybridization according to Affymetrix Mouse Exon Array 1.0ST protocol (Affymetrix, Santa Clara, US)
|
| Scan protocol |
Affymetrix Genechip 7G ScanMachinePrt1
|
| Description |
Transfection of murine bone-marrow derived macrophages (BMDMs) with non-targeting (control) siRNA and 11 sequence-specific siRNAs was performed using a cationic lipid transfection reagent (Lipofectamine2000) prior to stimulation with IFNγ. Total RNA was harvested from cells and gene expression measured on Affymetrix GeneChip Mouse Exon 1.0 ST Arrays.
|
| Data processing |
RMA normalization using "core" set of probes
|
| |
|
| Submission date |
Jan 23, 2009 |
| Last update date |
May 26, 2009 |
| Contact name |
Paul A Lacaze |
| Organization name |
University of Edinburgh
|
| Department |
School of Medicine
|
| Lab |
Division of Pathway Medicine
|
| Street address |
49 Little France Crescent
|
| City |
Edinburgh |
| ZIP/Postal code |
EH16 4SB |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL6096 |
| Series (1) |
| GSE14534 |
Combined genome-wide expression profiling and targeted RNA interference in primary mouse macrophages |
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