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Sample GSM602668 Query DataSets for GSM602668
Status Public on Nov 23, 2011
Title BMMC-pAPM-miR-221 vs BMMC-pAPM-miR-221m replicate 1
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 1 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.

Data table header descriptions
ID_REF
VALUE Normalized log10 ratio (Cy5/Cy3) representing test/reference

Data table
ID_REF VALUE
A_51_P244408 0.134
A_52_P94150 11.228
A_52_P336685 3.178
A_52_P58558 15.660
A_51_P374476 0.174
A_52_P267564 17.526
A_51_P328489 2.483
A_51_P508485 3.749
A_52_P302422 0.472
A_52_P558401 11.294
A_51_P421300 2.503
A_51_P467224 7.908
A_51_P450632 2.277
A_52_P18236 5.575
A_51_P142153 2.747
A_52_P515094 0.165
A_52_P531140 8.308
A_52_P403157 14.791
A_51_P515883 0.018
A_51_P388310 2.731

Total number of rows: 41174

Table truncated, full table size 762 Kbytes.




Supplementary file Size Download File type/resource
GSM602668_3_2_221_vs_3_2_221m.txt.gz 3.6 Mb (ftp)(http) TXT
Processed data included within Sample table

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