strain: Fisher 344 gender: male age: 4 weeks at the beginning of the experiment tissue: heart treatment: ultrafine particles (UFP) treatment duration: 3 months
Extracted molecule
total RNA
Extraction protocol
Surgical tissue was treated with 500ul of TriZol (Invitrogen, Carlsbad, CA) in a 1.5ml Eppendorf tube, and homogenized for 15 seconds. The homogenate was centrifuged for 20 min (4C) at 12,000 x g, and supernatant was transferred to a fresh Eppendorf tube and precipitated by the addition of 100ul of chloroform and vortexed vigorously for 15 seconds. The sample was incubated for 3 minutes at room temperature and centrifuged for 15 minutes (4C) at 10,000 x g. The aqueous phase was transferred to a fresh Eppendorf tube and precipitated by the addition of 250l isopropanol. The precipitate was incubated for 10 min at room temperature and centrifuged for 20 minutes (4C) at 10,000 x g. The sample was washed once with 0.7 ethanol and the pellet was airdried prior to resuspension in 100ul of DEPC treated water. The RNA was purified by RNeasy Clean Up column (Qiagen, Valencia, CA).
Label
biotin
Label protocol
cRNA synthesis and labeling was carried out by using Illumina Total Prep RNA Amplification Kit (Ambion, Austin, TX). Whole quantity of synthesized cDNA was added to 2.5ul T7 10x reaction buffer, 2.5ul biotinNTP mix, 2.5ul T7 Enzyme mix, and was incubated for 14 hrs at 37C followed by RNeasy column purification (Qiagen, Valencia, CA).
Hybridization protocol
Standard protocol as recommended by Illumina.
Scan protocol
Standard protocol as recommended by Illumina.
Description
Total RNA was extracted from the hearts of rats exposed for 3 months to filtered air (control) vs. coarse, fine and ultrafine particles.
Data processing
The data was preprocessed by the Bioconductor lumi package (version 1.5.17). It was vst transformed and quantile normalized.
