|
| Status |
Public on Mar 01, 2010 |
| Title |
MSC_undiff_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
bone marrow mesenchymal stem cells, undifferentiated
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: bone marrow
cell type: cultured mesenchymal stem cells (hMSC)
condition: undifferentiated
|
| Treatment protocol |
Cells grown in expansion medium.
|
| Growth protocol |
Human bone-marrow and skin biopsies were obtained from healthy donors. Mesenchymal stem cells (from bone marrow) and skin fibroblasts were isolated as described. All cells were cultured in expansion medium (Dulbecco’s modified Eagle’s medium (DMEM), with 4.5 g/l D-glucose supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, and 50 μg/ml gentamicin; all culture reagents from Invitrogen, Carlsbad, CA).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated by using the miRNeasy Mini Kit (Qiagen, Valencia, CA), according to manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
miRNA Microarray System Protocol (Agilent Technologies) was used to label RNA. Basically, 100 ng of total RNA were dephosphorylated and Cyanine 3-pCp molecule was ligated to the 3´ end of each RNA molecule by using T4 RNA ligase.
|
| |
|
| Hybridization protocol |
100 ng of Cy3 labelled RNA were hybridized to Human miRNA V2 Microarray 8x15K (G4470B, Agilent Technologies) for 20 hours at 55ºC in a hybridization oven (G2545A, Agilent) set to 15 rpm in a final concentration of 1X GE Blocking Agent and 1X Hi-RPM Hybridization Buffer, according to manufacturer's instructions (miRNA Microarray System Protocol, Agilent Technologies).
|
| Scan protocol |
Arrays were scanned at 5um resolution on an Agilent DNA Microarray Scanner (G2565BA, Agilent Technologies) using the default settings for miRNA Microarray v2.0 (miRNA Microarray System Protocol, Agilent Technologies). Images provided by the scanner were analyzed using Agilent´s software Feature Extraction version 9.5.3.1.
|
| Description |
miRNA expression in undifferentiated bone marrow-derived MSC.
|
| Data processing |
Data pre-processing and differential expression analysis were done using the Bioconductor package AgiMicroRna. The Total Gene Signal provided by the Agilent Feature Extracion image analysis software was used for the data analysis. Data were normalized between arrays using the quantile method. The image analysis software attach to each feature a flag that identifies different quantification errors of the signal that can be used to filter out the microRNAs that do not reach a minimum of quality. This filtering was done after the normalization of the Total Gene Signal. We kept for the analysis those microRNA genes flagged by Agilent Feature Extraction software as detected (gIsGeneDetected=1) in at least one experimental condition. Basically, the gIsGeneDetected filtering removes microRNAs that are not expressed in any experimental condition.
|
| |
|
| Submission date |
Nov 30, 2009 |
| Last update date |
Jan 22, 2010 |
| Contact name |
Manuel Angel Gonzalez |
| E-mail(s) |
magonzalez@cnic.es
|
| Organization name |
CNIC
|
| Street address |
Melchor Fernandez Almagro, 3
|
| City |
Madrid |
| ZIP/Postal code |
28032 |
| Country |
Spain |
| |
|
| Platform ID |
GPL8227 |
| Series (1) |
| GSE19232 |
miR-335 orchestrates cell proliferation, migration and differentiation in human mesenchymal stem cells |
|
