gender: male strain: Wistar post-instillation period: 365 days
Treatment protocol
0.2 mg or 0.4 mg of SWCNTs was suspended in 0.4 ml of distilled water including 0.1% Triton X-100. Each material suspension was intratracheally instilled once in rats. The animals were dissected at 3 days, 7 days, 30 days, 90 days, 180 days, 365 days, or 754 days after instillation.
Growth protocol
Nine-week-old male Wistar rats purchased from Hokudo Co., Ltd (Sapporo, Japan) were divided into groups (n = 6 per group per time point). SWCNTs were intratracheally administered into rats as a single injection (0.2 mg, or 0.4 mg SWCNTs per rat). The vehicle control groups received 0.1% Triton X-100 per rat. After intratracheal instillation treatment, rats were housed within polycarbonate cages at a controlled temperature of 22 ◦C with a chow diet ad libitum, and were dissected at 3 days, 7 days, 30 days, 90 days, 180 days, 365 days, and 754 days post-instillation. Lungs of anesthetized rats were perfused with physiological saline, excised, and used for the morphological observation, histopathological findings and comprehensive gene expression analysis microarray analysis. All procedures and animal handling were performed according to the guidelines described in the Japanese Guide for the Care and Use of Laboratory Animals as approved by the Animal Care and Use Committee, National Institute of Advanced Industrial Science and Technology, Japan.
Extracted molecule
total RNA
Extraction protocol
The right lungs (n = 4 per group per time point) were homogenized using QIAzol lysis reagent with a Tissue Ruptor (Qiagen, Tokyo, Japan). Total RNA from the homogenates was extracted using the RNeasy Midi kit (Qiagen) following the manufacturer’s instructions. RNA was quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, US) and the quality of the samples was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, US).
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 365 days in L-SWCNT-instilled in rat lungs
Data processing
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 014879_D_F_20110906). 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 (CN200_004: N=3).
