|
| Status |
Public on Oct 21, 2009 |
| Title |
EMG2 |
| Sample type |
RNA |
| |
|
| Source name |
EMG cell line -Dox (24 h)
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: Cell line with inducible expression of p33ING1
agent: uninduced (control)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared using TriReagent (Sigma), following the manufacturer's protocol.
|
| 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. Labelled RNA was purified using Micro Bio Spin 6 Columns (Bio-Rad, 732-6221).
|
| |
|
| Hybridization protocol |
100 ng of Cy3 labelled RNA were hybridized 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 v1.0 (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 |
no additional information
|
| Data processing |
Data files from Feature Extraction were imported into GeneSpring® GX software version 9.0. (Agilent Technologies). Quantile normalization was performed and log2 transformed expression values were obtained for each probe. Probes were also flagged (Present, Marginal, Absent) using GeneSpring® default settings. Probes were filtered for further analysis according to expression (all miRNAs which raw expression levels are within the range 20%-100% of expression in all replicates of at least one condition were retained) and flags (all miRNAs flagged as Present and Marginal in all replicates of at least one condition were retained). Expression ratios of Ing1-deficient vs wild-type mouse MEFs and Doxycycline-induced EMG cells vs uninduced controls were calculated and transformed into a log2 scale
|
| |
|
| Submission date |
Oct 20, 2009 |
| Last update date |
Oct 21, 2009 |
| Contact name |
Sergio Callejas |
| Organization name |
Centro Nacional de Investigaciones Cardiovasculares (CNIC)
|
| Department |
Genomics Unit
|
| Lab |
Genomics Unit
|
| Street address |
Melchor Fernández Almagro, 3
|
| City |
Madrid |
| ZIP/Postal code |
28029 |
| Country |
Spain |
| |
|
| Platform ID |
GPL9081 |
| Series (1) |
| GSE18645 |
Regulation of the microRNA processor DGCR8 by the tumor suppressor ING1 |
|
