|
| Status |
Public on Aug 20, 2008 |
| Title |
miR181b/control B cell exp1 replicate1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
primary spleen B cell-tranduced with control-GFP vector
|
| Organism |
Mus musculus |
| Characteristics |
strain: C 57 BL/6
|
| Treatment protocol |
Mouse primary B cells were transduced with retroviral supernatants for 20 hours in the presence of 8 ug/ml polybrene (Sigma), 25ug/ml LPS (Sigma) and 10ng/ml IL-4 (Peprotech). GFP+ cells were isolated by cell sorting 2 days after transduction.
|
| Growth protocol |
Mouse primary B cells were purified from spleens of C57/BL6 mice by anti-CD43 immunomagnetic depletion (Miltenyi Biotech) and cultured in 25ug/ml LPS (Sigma), 10ng/ml IL-4 (Peprotech), 10mM Hepes (Gibco), 50uM b-mercaptoethanol (Gibco) 10% FCS RPMI medium
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with Trizol (Invitrogen). Commercial RNeasy kit (Qiagen) was used in spin-column format following manufacturer instructions. Total RNA was analysed by Lab-chip technology on an Agilent 2100 Bioanalyzer. Samples' RNA Integrity Numbers were in the range 9.7 to 9.8
|
| Label |
Cy3
|
| Label protocol |
Amount of nucleic acid labeled: 2ug.Amplification: by RNA polymerases.Commercial "Two-Color Microarray-Based Gene Expression Analysis" kit by following manufacturer instructions. Agilent manual G4140-90050 Ver. 5.0 Aug 2006. Briefly, MMLV-RT retrotranscription of sample from a T7 promoter primer is followed by a T7 RNA pol catalysed in vitro transcription reaction in the presence of either Cy3-CTP or Cy5-CTP fluorophores. Labeled samples are purified with silica-based RNeasy spin columns (Qiagen).
|
| |
|
| Channel 2 |
| Source name |
primary spleen B cell-tranduced with miR181b-GFP vector
|
| Organism |
Mus musculus |
| Characteristics |
strain: C 57 BL/6
|
| Treatment protocol |
Mouse primary B cells were transduced with retroviral supernatants for 20 hours in the presence of 8 ug/ml polybrene (Sigma), 25ug/ml LPS (Sigma) and 10ng/ml IL-4 (Peprotech). GFP+ cells were isolated by cell sorting 2 days after transduction.
|
| Growth protocol |
Mouse primary B cells were purified from spleens of C57/BL6 mice by anti-CD43 immunomagnetic depletion (Miltenyi Biotech) and cultured in 25ug/ml LPS (Sigma), 10ng/ml IL-4 (Peprotech), 10mM Hepes (Gibco), 50uM b-mercaptoethanol (Gibco) 10% FCS RPMI medium
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with Trizol (Invitrogen). Commercial RNeasy kit (Qiagen) was used in spin-column format following manufacturer instructions. Total RNA was analysed by Lab-chip technology on an Agilent 2100 Bioanalyzer. Samples' RNA Integrity Numbers were in the range 9.7 to 9.8
|
| Label |
CY5
|
| Label protocol |
Amount of nucleic acid labeled: 2ug.Amplification: by RNA polymerases.Commercial "Two-Color Microarray-Based Gene Expression Analysis" kit by following manufacturer instructions. Agilent manual G4140-90050 Ver. 5.0 Aug 2006. Briefly, MMLV-RT retrotranscription of sample from a T7 promoter primer is followed by a T7 RNA pol catalysed in vitro transcription reaction in the presence of either Cy3-CTP or Cy5-CTP fluorophores. Labeled samples are purified with silica-based RNeasy spin columns (Qiagen).
|
| |
|
| |
| Hybridization protocol |
Chamber type: SureHyb hybridization chamber (Agilent). Quantity of labeled extract used: 825 ng. Duration: 17 hours.Volume: 100 uL.Temperature (ºC): 65.
|
| Scan protocol |
Scanned on an G2565BA DNA microarray scanner (Agilent). Images were quantified using Agilent Feature Extraction Software (ver. 9.1.3.1).
|
| Description |
Biolgical replicate 1 of 1. Primary spleen B cell-tranduced with control-GFP and miR181b vectors selected by cell sorting after 2 days in LPS+IL-4 cultures
|
| Data processing |
Linear Lowess normalization performed with Agilent's Feature Extraction software.
|
| |
|
| Submission date |
Jul 17, 2008 |
| Last update date |
Aug 20, 2008 |
| Contact name |
Almudena R. Ramiro |
| E-mail(s) |
arodriguezr@cnio.es
|
| URL |
http://www.cnio.es/es/grupos/plantillas/presentacion.asp?grupo=50004655
|
| Organization name |
CNIO
|
| Department |
Molecular Oncology
|
| Lab |
DNA Hypermutation and Cancer Group
|
| Street address |
Calle Melchor Fernandez Almagro 3
|
| City |
Madrid |
| State/province |
Madrid |
| ZIP/Postal code |
28029 |
| Country |
Spain |
| |
|
| Platform ID |
GPL4134 |
| Series (2) |
| GSE12158 |
Murine spleen B cells miR181b vs control (G4122F) |
| GSE12186 |
microRNA expression profile of activated primary B cells and miR181b overexpressing primary B cells |
|
