|
| Status |
Public on May 28, 2014 |
| Title |
miRNA_2.5 % FCS_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
ASM, 24hr
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Airway Smooth Muscle
|
| Growth protocol |
Confluent cells were growth-arrested by FCS deprivation for 24 h before being treated with dexamamethasone for 60 min, and then stimulated with FCS for a further 24 h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the mirVana™miRNA isolation kit (Ambion Europe). RNA was eluted in 50 μl RNase-free water (Promega UK, Southampton, UK) and stored at -80 °C. RNA content and purity was measured using a BioTek PowerWave XS (SSi Robotics, Tustin, CA, U.S.A.) spectrophotometer.
|
| Label |
Cy3
|
| Label protocol |
miRNA samples were labeled using cy3
|
| |
|
| Hybridization protocol |
miRNA-enriched RNA samples (100 ng) used in the microRNA microarrays were labeled with the Labeling Spike-In solution using Agilent’s miRNA Complete Labeling and Hyb Kit and then the samples were dephosphorylated by incubation at 37 oC on a heat block for 30 min. DMSO was added to the dephosphorylated RNA for denaturation for 10 min, followed by the assembly of the Labeling Reaction, where samples were incubated with Cyanine 3-pCp and allowed to ligate for 2 h at 16 oC. The labeled RNA was purified using Micro Bio-Spin 6 columns (Bio-Rad) and the desalted samples were dried up in a vacuum concentrator at 50 oC for 1 h to remove DMSO. Samples were resuspended in Hybridization Mix using Hyb Spike-In solution and Hybridization Buffer and were loaded onto Agilent Human miRNA microarrays and hybridized at 55 oC for 20 h at 20 rpm using Agilent’s Hybridization oven and SureHyb chamber. The microarrays were then disassembled and washed in GE Wash Buffer 1 (Agilent) for 60 sec at RT, followed by GE Wash Buffer 2 for 60 sec at 37 oC. The microarrays were scanned with the Agilent Microarray Scanner G2565BA using the profile for miRNA microarrays (AgilentHD_miRNA) at 2 μm resolution, dye channel Green, Scan Area 61 x 21.6 mm.
|
| Scan protocol |
Scanned on an Agilent Sureprint G3 scanner.
|
| Description |
Gene expression after 24h
|
| Data processing |
Agilent Feature Extraction Software
|
| |
|
| Submission date |
May 16, 2014 |
| Last update date |
May 28, 2014 |
| Contact name |
Mark Perry |
| E-mail(s) |
m.perry@imperial.ac.uk
|
| Organization name |
NHLI, Imperial College London
|
| Department |
Airway Disease
|
| Street address |
Dovehouse Street
|
| City |
London |
| ZIP/Postal code |
SW36LY |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL15018 |
| Series (2) |
| GSE57765 |
Gene expression analysis of stimulated primary ASM cells [microRNA] |
| GSE57766 |
Gene expression analysis of stimulated primary ASM cells |
|
