|
| Status |
Public on May 28, 2014 |
| Title |
2.5 % FCS_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
Human primary airway smooth muscle cultured for 24 h
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Airway Smooth Muscle
|
| Growth protocol |
Confluent cells were growth-arrested by FCS deprivation for 24 h in DMEM supplemented with sodium pyruvate (1 mM), L-glutamine (2 mM), nonessential amino acids (1:100), penicillin (100 U/ml)/streptomycin (100 μg/ml), amphotericin B (1.5 μg/ml), and bovine serum albumin (0.1 %). Cells were stimulated with 2.5 % FCS for 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 |
mRNA samples were dual labeled using either cy3 or cy5
|
| |
|
| Hybridization protocol |
Total RNA samples (50 ng) used in lncRNA and mRNA microarrays were initially labeled with Spike-In control A (for Cyanine 3-CTP) or B (for Cyanine 5-CTP). Labelled samples were then used for cDNA synthesis using the cDNA Master Mix (Agilent) and were incubated for 2 h at 40 oC followed by 15 min at 70 oC to inactivate the AffinityScript enzyme. The synthesized cDNA was then used for cRNA synthesis and amplification using the Transcription Master Mix with either Cyanine 3 or Cyanine 5 and incubated at 40 oC for 2 h. Labeled and amplified RNA was then purified using the RNeasy Mini Kit (Qiagen) and quantified using the NanoDrop Spectrophotometer’s Microarray Measurement function. The Cyanine 3 or Cyanine 5 concentrations and the cRNA concentration were used to calculate the yield (μg) and the specific activity (pmol Cy3 or Cy5 per μg cRNA) of each sample. For each microarray reaction, 300 ng of Cyanine 3- and 300 ng of Cyanine 5-labelled, linearly amplified cRNA samples were mixed and incubated together with Fragmentation buffer for 30 min, followed by the addition of Hybridization buffer. Samples were loaded onto SurePrint G3 (8 x 60 K) microarray slides (Agilent) and hybridized at 65 oC for 17 h at 10 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, followed by an acetonitrile wash (10 sec at RT) and the final wash in Stabilization and Drying solution for 30 sec at RT, to improve microarray results by preventing ozone-mediated fluorescent signal degradation. The microarrays were scanned with the Agilent Microarray Scanner G2565BA using the profile for 2-colour microarrays (AgilentG3_GX_2Color) at 5 μm resolution, dyes channel Red&Green, Scan Area 61 x 21.6 mm.
|
| Scan protocol |
Scanned on an Agilent Sureprint G3 scanner.
|
| Description |
Raw data: US91903684_252800413362_S01_GE2_105_Dec08_1_3.txt (Cy3)
Sample 3 OX125 FCS
|
| 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 |
GPL13607 |
| Series (2) |
| GSE57764 |
Gene expression analysis of stimulated primary ASM cells [mRNA] |
| GSE57766 |
Gene expression analysis of stimulated primary ASM cells |
|
