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Status |
Public on Dec 13, 2010 |
Title |
Control Lung 20 (miRNA) |
Sample type |
RNA |
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Source name |
Total RNA enriched with small RNA species from mouse lungs labeled with Cyanine-5 (red)
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Organism |
Mus musculus |
Characteristics |
tissue: part of lung gender: female age: 11-13 weeks strain: C57BL/7 date of exposure: 2008-11-02 exposure: exposed to filtered air for 1 h/day for 11 consecutive days and were sacrificed 5 days following the last exposure. time post exposure: 5 days set: Hybridization Block 2
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Treatment protocol |
Briefly, fortyfive time-mated, nulliparous, young adult female mice at GD 3 were housed in cages under controlled environmental conditions with 12 h light–dark cycles. Food and tap water were provided ad libitum. Exposure was performed by whole-body inhalation to 42.4 ± 2.9 (SEM) mg nanoTiO2/m3 or to the filtered air for 1 h/day from GD 8–18. Seventeen of the time-mated mice (“NP3 females” in Hougaard et. al, submitted) that had not conceived, as evidenced by lack of pups and absence of interuterine implantation sites were used in the present study as a group of adult females. Animals were sacrificed by cardiac puncture five days after the last exposure. The lungs and livers were dissected, weighed, and snap frozen. Samples were stored at -80°C until analysis. All procedures complied with EC Directive 86/609/EEC and Danish law regulating experiments on animals (permission 2006/561-1123).
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Extracted molecule |
total RNA |
Extraction protocol |
The mirVana miRNA Isolation Kit (Ambion, Streetsville, ON, Canada) was used to prepare total RNA enriched with small RNA species from randomly selected left lung sections. All RNA samples showing A260/280 ratios between 2.0 and 2.1 were further analysed by Agilent 2100 Bioanalyzer (Agilent Technologies Inc.,) to determine RNA integrity. Only high quality RNA (28S/18S > 1.8) was used for further analysis.
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Label |
Cy3
|
Label protocol |
Freshly isolated individual total lung RNA samples from 8 controls and 8 treated groups were labelled using the Agilent miRNA Complete Labelling and Hybridisation Kit (Agilent Technologies, Inc.,). Briefly, 100 ng of total RNA was dephosphorylated by incubation with calf intestinal phosphatase at 37ºC for 30 min, denatured using 100 % DMSO at 100ºC for 5 min, then labelled with pCp-Cy3 using T4 ligase by incubation at 16ºC for 1 hour.
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Hybridization protocol |
The labelled RNA samples were hybridised to an individual array on 8 x 15K format Agilent mouse miRNA array slides. Hybridizations were performed in SureHyb chambers (Agilent) at 55ºC for 20 hours.
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Scan protocol |
Arrays were washed, scanned at a resolution of 5 m using an Agilent G2505B scanner and data were acquired using Agilent Feature Extraction software version 9.5.3.1.
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Description |
Total RNA enriched with small RNA species
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Data processing |
Non-background subtracted raw data were quantile normalized (Bolstad et al. 2003). Present calls were determined as signals that were greater than 3 trimmed SDs above the trimmed mean of the the (-)3xSLv1 probes on the array. Probes with technical replicates for a miRNA were averaged using the median signal intensity.
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Submission date |
Dec 24, 2009 |
Last update date |
Dec 13, 2010 |
Contact name |
Andrew Williams |
Organization name |
Health Canada
|
Street address |
50 Columbine
|
City |
Ottawa |
State/province |
ON |
ZIP/Postal code |
K1A OK9 |
Country |
Canada |
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|
Platform ID |
GPL8824 |
Series (1) |
GSE19487 |
Lungs of mice exposed to subtoxic doses of nano-titanium dioxide particles by inhalation |
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