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Sample GSM1388210 Query DataSets for GSM1388210
Status Public on May 28, 2014
Title 2.5 % FCS_rep2
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 cy5
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_252800413360_S01_GE2_105_Dec08_1_3.txt (Cy5)
Sample 2 OX120 FCS
Data processing Agilent Feature Extraction Software
Submission date May 16, 2014
Last update date May 28, 2014
Contact name Mark Perry
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

Data table header descriptions
VALUE normalized signal

Data table
1 8.56E+04
2 9.15E+00
3 6.07E+00
4 2.25E+02
5 3.43E+02
6 2.95E+01
7 2.78E+03
8 1.77E+03
9 1.25E+01
10 3.52E+01
11 1.16E+01
12 2.01E+02
13 6.64E+02
14 5.08E+02
15 3.64E+03
16 2.13E+01
17 4.40E+01
18 1.23E+01
19 7.15E+00
20 5.19E+02

Total number of rows: 62976

Table truncated, full table size 911 Kbytes.

Supplementary data files not provided
Processed data included within Sample table

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