|
| Status |
Public on Dec 01, 2020 |
| Title |
Lung_403_Control_Day3_rep4 |
| Sample type |
RNA |
| |
|
| Source name |
Rat, Lung, NM-403, Control, Day 3
|
| Organism |
Rattus norvegicus |
| Characteristics |
strain: Sprague Dawley
tissue: accessory lobe of lung
gender: female
age: 13 weeks-old (at the onset of inhalation)
treatment: control
dose: Control
time: Day 3
|
| Treatment protocol |
13 week old female Sprague Dawley rats were exposed by nose-only inhalation 6 hours/day, 5 days/week for 4 weeks to two doses of NM-403 carbon nanotube.
|
| Growth protocol |
Accessory lobe of rat lungs were stored in RNA later at -20°C before use
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA were disrupted using gentleMACS™ Dissociator (Miltenyi Biotech) and extracted from accessory lobe of lungs with Nucleospin® RNA midi kit (Macherey-Nagel) according to the manufacturer’s instructions. RNA quality was determined by spectrophotometry (A260nm/A280nm > 1.8) and by capillary electrophoresis using Agilent 2100 Bioanalyser (RIN > 7, Agilent RNA 6000 NanoRNA 6000 Nano® Kit, Santa Clara, CA, USA).
|
| Label |
Cy3
|
| Label protocol |
For each of the 72 samples (6 control, 6 exposed rats to low dose and 6 exposed rats to high dose per time group), 100 ng of RNA were labelled with Cyanine 3-CTP using Low Input Quick Amp Labelling kits (One-Color Microarray-Based Gene Expression Analysis, version 6.7, Agilent Technologies) and purified (RNeasy Mini kit, QIAGEN) according to the manufacturer's instruction . Dye incorporation and cRNA yield and quality were checked by spectrophotometry and by electrophoresis (Agilent 2100 Bioanalyser).
|
| |
|
| Hybridization protocol |
600 ng of Cy3-CTP-labelled cRNA (Specific Activity > 10 pmol Cy3 per µg cRNA) were fragmented at 60°C for 30 min in a reaction volume of 25 µL containing 25X Agilent Fragmentation Buffer and 10X Agilent Blocking Agent following the manufacturer’s instructions. On cRNA fragmentation completion, 25 µL of 2X Agilent Hybridization Buffer were added to the fragmentation mixture. The subsequent purified labelled cRNAs were hybridized to Agilent G4853A SurePrint G3 Rat v2 GE 8*60K microarrays (Agilent Technologies) at 65°C overnight (17h) in a rotating hybridization oven. After hybridization, microarrays were washed 1 min at room temperature with Agilent Gene Expression Wash Buffer 1 and 1 min with 37°C Agilent Gene Expression Wash buffer 2.
|
| Scan protocol |
Slides were scanned immediately after washing using Agilent high-resolution scanner (G2505C) by setting: (i) one color scan channel for 8x60k array slides, (ii) scan area of 61x21.6 mm, (iii) scan resolution of 3 µm, (iv) dye channel to Green, (v) Tiff file dynamic range of 20 bits and (vi) Green PMT to 100 %.
|
| Description |
Gene expression in lung of control rat (filterd air) 3 days post exposure
Rat, Lung, NM-403, Control, Day 3, replicate 4
|
| Data processing |
Agilent Feature Extraction Software (v 11.0.1.1) was used for background subtraction and data were normalized using the GeneSpring software (v 14.9)
Data were normalized by shift to 75.0 percentile and filtered by flags and by expression based on the signal intensity values (to remove very low signal values and those that have reached saturation)
|
| |
|
| Submission date |
Apr 23, 2018 |
| Last update date |
Dec 01, 2020 |
| Contact name |
Carole Seidel |
| E-mail(s) |
[email protected]
|
| Organization name |
INRS
|
| Street address |
Rue du Morvan
|
| City |
Vandoeuvre les Nancy |
| ZIP/Postal code |
54519 |
| Country |
France |
| |
|
| Platform ID |
GPL22740 |
| Series (2) |
| GSE113531 |
Inhaled multi-walled carbon nanotubes-induced gene expression profile in rat lung (NM-403) |
| GSE113532 |
Inhaled multi-walled carbon nanotubes-induced gene expression profile in rat lung |
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