|
| Status |
Public on Sep 25, 2012 |
| Title |
A549_48hr_CNT-1 |
| Sample type |
RNA |
| |
|
| Source name |
Alveolar epithelial cell, 48hr, CNT-1, replicate 4
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Alveolar epithelial cell
cell line: A549
treatment: CNT-1
time: 48h
|
| Treatment protocol |
Each medium were replaced by three kinds of single-wall carbon nanotubes (SWCNTs), CNT-1, CNT-2 or CNT-3 in a 10-fold dilution series. Cell concentrations at this point were approximately 1.3 x 10^5 cells/mL. The cultures were incubated for 24 or 48 hours at 37 ºC in a humidified incubator with 5% CO2.
|
| Growth protocol |
Human alveolar epithelial cell line A549 were seeded into each well of a 96-well plate (Becton, Dickinson and Company, Franklin Lakes, NJ) and grown in the DMEM with 10% heat-inactivated fetal bovine serum medium for 24 hours.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from the cells using the QIAcube instrument (Qiagen, Tokyo, Japan) following the manufactures instructions. 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 Human Genome Oligo Microarrays (G4112F) 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 48hr in CNT-1-exposed human alveolar epithelial cell
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1-v1_Sep09 and Grid: 026652_D_F_20110819). 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. VALUE: Normalized signal intensity (log2)
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| |
|
| Submission date |
Sep 24, 2012 |
| Last update date |
Sep 25, 2012 |
| 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 |
Onogawa 16-1
|
| City |
Tsukuba |
| ZIP/Postal code |
3058569 |
| Country |
Japan |
| |
|
| Platform ID |
GPL13497 |
| Series (1) |
| GSE41101 |
Gene expression profiles in human alveolar epithelial cell line exposure to impurity-free single-wall carbon nanotubes |
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