|
| Status |
Public on Feb 02, 2015 |
| Title |
NM-401_1_162 [686] |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
NM-401
|
| Organism |
Mus musculus |
| Characteristics |
sampleID: 686
time point (days): 1
dose: 162
particle: NM-401
strain: C57BL/6
gender: female
|
| Treatment protocol |
The C57BL/6 mice were exposed in groups of 6 to 162 ug of NRCWE-26 or NM-401 via intratracheal instillation. In brief, mice were anesthetised with 4 % isoflurane until fully relaxed and 2.5 % during the instillation The mice were kept on their back at a 40-degree angle on a hot plate (37°C) during the entire procedure. 162 μg of NRCWE-26 or NM-401 was administered via a single intratracheal instillation. A 40 μl suspension was instilled followed by 150 μl air with a 250 μl SGE glass syringe. Control animals were instilled with vehicle (NanoPure water with 2% serum). After the instillation catheter was removed, breathing was observed in order to assure that the delivered material did not block the airways.
|
| Growth protocol |
Female C57BL/6 mice at the age of 5–7 weeks were obtained from Taconic (Ry, Denmark) and were allowed to acclimatise for 1-3 weeks before the experiment. All mice were fed on Altromin (no. 1324, Christian Petersen, Denmark) and had access to water ad libitum during the whole experiment. The mice were housed in groups of 10 animals in polypropylene cages with sawdust bedding and enrichment at controlled temperature 21±1°C and humidity 50±10% with a 12-h light/12-h dark cycle. Experiments were initiated at 8 weeks of age. All procedure complied with EC Directive 86/609/EEC and Danish laws regulating experiments on animals (permit 2010/561-1779). Mice did not display any signs of respiratory distress, lethargy or any other physical symptoms following exposure.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
At 1, 3 or 28 days post-instillation, the mice were anaesthetized as described above and killed by exsanguination via intracardiac puncture. Immediately after withdrawing the heart blood, the liver tissue were snap frozen in cryotubes in liquid N2 and stored at –80°C. Total RNA was extracted (n=6 per group) using Trizol reagent and further purified using the RNeasy Mini Kit with an on-column DNase I digestion according to the manufacturer's instructions. Total RNA concentration and quality was determined using a Nanodrop 1000 Spectrophotometer and an Agilent 2100 Bioanalyzer, respectively. RNA samples used for gene expression microarray had A260/280 ratios between 2.0 and 2.15 and RINs above 7.0.
|
| Label |
Cy5
|
| Label protocol |
Total RNA (200ng per array) was labeled using the Agilent Quick Amp Labeling Kit, Two-Color (Agilent Cat #: 5190-0444), according to the manufacturer's instructions. Control and experimental samples were labeled with Cyanine 5-CTP; whereas universal mouse reference RNA was labeled with Cyanine 3-CTP.
|
| |
|
| Channel 2 |
| Source name |
Universal Mouse Reference RNA (UMRR, Stratagene, Mississauga, ON, Canada)
|
| Organism |
Mus musculus |
| Characteristics |
reference: Universal Mouse Reference RNA (UMRR, Stratagene, Mississauga, ON, Canada)
|
| Treatment protocol |
The C57BL/6 mice were exposed in groups of 6 to 162 ug of NRCWE-26 or NM-401 via intratracheal instillation. In brief, mice were anesthetised with 4 % isoflurane until fully relaxed and 2.5 % during the instillation The mice were kept on their back at a 40-degree angle on a hot plate (37°C) during the entire procedure. 162 μg of NRCWE-26 or NM-401 was administered via a single intratracheal instillation. A 40 μl suspension was instilled followed by 150 μl air with a 250 μl SGE glass syringe. Control animals were instilled with vehicle (NanoPure water with 2% serum). After the instillation catheter was removed, breathing was observed in order to assure that the delivered material did not block the airways.
|
| Growth protocol |
Female C57BL/6 mice at the age of 5–7 weeks were obtained from Taconic (Ry, Denmark) and were allowed to acclimatise for 1-3 weeks before the experiment. All mice were fed on Altromin (no. 1324, Christian Petersen, Denmark) and had access to water ad libitum during the whole experiment. The mice were housed in groups of 10 animals in polypropylene cages with sawdust bedding and enrichment at controlled temperature 21±1°C and humidity 50±10% with a 12-h light/12-h dark cycle. Experiments were initiated at 8 weeks of age. All procedure complied with EC Directive 86/609/EEC and Danish laws regulating experiments on animals (permit 2010/561-1779). Mice did not display any signs of respiratory distress, lethargy or any other physical symptoms following exposure.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
At 1, 3 or 28 days post-instillation, the mice were anaesthetized as described above and killed by exsanguination via intracardiac puncture. Immediately after withdrawing the heart blood, the liver tissue were snap frozen in cryotubes in liquid N2 and stored at –80°C. Total RNA was extracted (n=6 per group) using Trizol reagent and further purified using the RNeasy Mini Kit with an on-column DNase I digestion according to the manufacturer's instructions. Total RNA concentration and quality was determined using a Nanodrop 1000 Spectrophotometer and an Agilent 2100 Bioanalyzer, respectively. RNA samples used for gene expression microarray had A260/280 ratios between 2.0 and 2.15 and RINs above 7.0.
|
| Label |
Cy3
|
| Label protocol |
Total RNA (200ng per array) was labeled using the Agilent Quick Amp Labeling Kit, Two-Color (Agilent Cat #: 5190-0444), according to the manufacturer's instructions. Control and experimental samples were labeled with Cyanine 5-CTP; whereas universal mouse reference RNA was labeled with Cyanine 3-CTP.
|
| |
|
| |
| Hybridization protocol |
Equal amounts of Cy5-labeled experimental cRNA and Cy3-labeled reference RNA (300ng) were co-hybridized to Agilent Sureprint G3 Mouse GE 8x60K microarray slides, according to the instructions outlined in the Agilent Two-Color Microarray-Based Gene Expression Analysis (Quick Amp Labeling) Protocol. Samples were hybridized for 17 hours at 65oC and 10 rpm.
|
| Scan protocol |
Scanned on an Agilent G2565AA scanner.
|
| Data processing |
Images were quantified using Agilent Feature Extraction Software (version A.8.5.1.1). Non background subtracted median signal intensities were LOWESS normalized using the maanova library in R. Probes with technical replicates were averaged using the median.
|
| |
|
| Submission date |
Sep 12, 2014 |
| Last update date |
Feb 02, 2015 |
| Contact name |
Sarah Søs Poulsen |
| E-mail(s) |
spo@nrcwe.dk
|
| Phone |
4539165225
|
| Organization name |
The National Research Centre for the Working Environment
|
| Street address |
Knud den Stores Vej 39B, 2.MF
|
| City |
Roskilde |
| State/province |
Denmark |
| ZIP/Postal code |
4000 |
| Country |
Denmark |
| |
|
| Platform ID |
GPL10787 |
| Series (1) |
| GSE61366 |
Changes in cholesterol homeostasis and acute phase response following pulmonary exposure to multi-walled carbon nanotubes links carbon nanotube exposure to risk of cardiovascular disease |
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