|
| Status |
Public on Sep 11, 2014 |
| Title |
CNT-1_NR8383 |
| Sample type |
RNA |
| |
|
| Source name |
Rat alveolar macrophages, 24hr, CNT-1, replicate 4
|
| Organism |
Rattus norvegicus |
| Characteristics |
cell type: alveolar macrophages
cell line: NR8383
treatment: CNT-1
time: 24h
|
| Treatment protocol |
The culture medium were replaced by two kinds of the working solutions including impurity-free single-wall carbon nanotubes (CNT-1 or CNT-2), respectively. These working solutions were dissolved in 1mg/mL bovine serum albumin (BSA). Cell concentrations at this point were approximately 2.0 x 10^5 cells/mL. The cultures were incubated for 24 hours at 37 ºC in a humidified incubator with 5% CO2.
|
| Growth protocol |
Rat alveolar macrophages NR8383 were seeded into each well of a 96-well plate and grown in the F-12K with 15% heat-inactivated fetal bovine serum medium for 24 hours.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After 24 hours, total RNA was extracted from the cells using the RNeasy mini (Qiagen, Tokyo, Japan) according to the manufactures instructions. Total RNA was quantified using a NanoDrop-2000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA) and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Tokyo, Japan). Dye incorporation and cRNA yield were checked with the NanoDrop ND-2000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Rat Genome Oligo Microarrays (G4131F) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent).
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 4x44k array slides (Scan resolution 5um).
|
| Description |
Gene expression after 24hr in CNT-1-exposed rat alveolar macrophages
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1_107_Sep09). Background detrend: On (Feat NCRange, LoPass). Multiplicative detrend: True. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. Normalized data is the average of n=4.
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| |
|
| Submission date |
Sep 10, 2014 |
| Last update date |
Sep 11, 2014 |
| Contact name |
Katsuhide Fujita |
| E-mail(s) |
ka-fujita@aist.go.jp
|
| Organization name |
National Institute of Advanced Industrial Science and Technology
|
| Department |
Research Institute of Science for Safety and Sustainability
|
| Street address |
16-1 Onogawa
|
| City |
Tsukuba |
| ZIP/Postal code |
3058569 |
| Country |
Japan |
| |
|
| Platform ID |
GPL7294 |
| Series (1) |
| GSE61319 |
Gene expression profiles in rat alveolar macrophages exposure to impurity-free single-wall carbon nanotubes |
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