|
| Status |
Public on Nov 23, 2011 |
| Title |
BMMC-pAPM-miR-221 vs BMMC-pAPM-miR-221m replicate 3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
BMMC transduced with pAPM-miR-221
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Bone marrow-derived mast cells
Sex: Female
strain: C57Bl/6
expression: wild type miR-221
|
| Treatment protocol |
BMMCs were transduced with pAPM-miR-221 or pAPM-miR-221m lentiviruses, selected with 2mg/mL of puromycin for 48 hours and expanded previous to RNA isolation.
|
| Growth protocol |
Bone marrow cells from C57Bl/6 female mice were differentiated and maintained in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2mM L-glutamine, 100U/mL penicillin, 100μg/mL streptomycin, 0.1mM non essential amino acids, 50μM β-mercaptoethanol and 50% WEHI-3 conditioned supernatant as a source of IL-3. Three weeks after differentiation the population was homogeneous, with ~95% of the cells in culture being c-Kit+ and FceRI+ at surface staining and testing positive for toluidine blue staining.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer’s instructions. The quality of extracted RNA was analyzed using the Agilent 2100 Bioanalyzer. Samples used for the experiment had RNA integrity numbers (RIN) between 8.5 and 9.3.
|
| Label |
Cy5
|
| Label protocol |
For the linear T7-based amplification step, 1 μg of each total RNA sample was used as starting material. To produce Cy3- and Cy5-labeled cRNA, the RNA samples were amplified and labeled using the Agilent Low RNA Input Linear Amp Kit (Agilent Technologies) following the manufacturer’s protocol. Yields of cRNA and the dye-incorporation rate were measured with the ND-1000 Spectrophotometer (NanoDrop Technologies). Control samples miR-221m were labeled with Cy3 and experimental samples miR-221 were labeled with Cy5.
|
| |
|
| Channel 2 |
| Source name |
BMMC transduced with pAPM-miR-221m
|
| Organism |
Mus musculus |
| Characteristics |
Sex: Female
strain: C57Bl/6
cell type: Bone marrow-derived mast cells
expression: mutant miR-221
|
| Treatment protocol |
BMMCs were transduced with pAPM-miR-221 or pAPM-miR-221m lentiviruses, selected with 2mg/mL of puromycin for 48 hours and expanded previous to RNA isolation.
|
| Growth protocol |
Bone marrow cells from C57Bl/6 female mice were differentiated and maintained in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2mM L-glutamine, 100U/mL penicillin, 100μg/mL streptomycin, 0.1mM non essential amino acids, 50μM β-mercaptoethanol and 50% WEHI-3 conditioned supernatant as a source of IL-3. Three weeks after differentiation the population was homogeneous, with ~95% of the cells in culture being c-Kit+ and FceRI+ at surface staining and testing positive for toluidine blue staining.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer’s instructions. The quality of extracted RNA was analyzed using the Agilent 2100 Bioanalyzer. Samples used for the experiment had RNA integrity numbers (RIN) between 8.5 and 9.3.
|
| Label |
Cy3
|
| Label protocol |
For the linear T7-based amplification step, 1 μg of each total RNA sample was used as starting material. To produce Cy3- and Cy5-labeled cRNA, the RNA samples were amplified and labeled using the Agilent Low RNA Input Linear Amp Kit (Agilent Technologies) following the manufacturer’s protocol. Yields of cRNA and the dye-incorporation rate were measured with the ND-1000 Spectrophotometer (NanoDrop Technologies). Control samples miR-221m were labeled with Cy3 and experimental samples miR-221 were labeled with Cy5.
|
| |
|
| |
| Hybridization protocol |
The hybridization procedure was performed according to the Agilent 60-mer oligo microarray processing protocol using the Agilent Gene Expression Hybridization Kit (Agilent Technologies). Briefly, 825 ng of the corresponding Cy3- and Cy5-labeled fragmented cRNA were combined and hybridized overnight (17 hours, 65 °C) to Agilent Whole Mouse Genome Oligo Microarrays 4x44K using Agilent’s recommended hybridization chamber and oven. Finally, the microarrays were washed once with 6x SSPE buffer containing 0.005% N-lauroylsarcosine for 1 min at room temperature followed by a second wash with pre-heated 0.06x SSPE buffer (37 °C) containing 0.005% N-lauroylsarcosine for 1 min. The last washing step was performed with acetonitrile for 30 sec.
|
| Scan protocol |
Fluorescence signals of the hybridized Agilent Oligo Microarrays were detected using Agilent’s DNA microarray scanner (Agilent Technologies).
|
| Description |
Biological replicate 3 of 3. Cells overexpressing miR-221 versus control cells overexpressing miR-221m.
|
| Data processing |
The Agilent Feature Extraction Software (FES) was used to read out and process the microarray image files. For determination of differential gene expression FES derived output data files were further analyzed using the Rosetta Resolver gene expression data analysis system (Rosetta Biosoftware).
|
| |
|
| Submission date |
Sep 30, 2010 |
| Last update date |
Nov 23, 2011 |
| Contact name |
Silvia Monticelli |
| E-mail(s) |
silvia.monticelli@irb.unisi.ch
|
| URL |
http://www.irb.ch
|
| Organization name |
Institute for Research in Biomedicine
|
| Lab |
Molecular Immunology
|
| Street address |
Via Vincenzo Vela, 6
|
| City |
Bellinzona |
| ZIP/Postal code |
6500 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL7202 |
| Series (1) |
| GSE24462 |
Murine bone marrow-derived mast cells (BMMCs) transduced with pAPM lentiviruses overexpressing miR-221 vs control overexpressing miR-221m. |
|
