cell line: U251 astrocyte cell line infection: control time: 6 hpi
Treatment protocol
U251 cells were inoculated with H5N1 at MOI 1.0 or not infected. After 40 min adsorption, cells were washed once using warm phosphate-buffered saline (PBS) and then incubated in DMEM containing 0.2% FBS at 37℃. At time points 6, 12, and 24 hpi, total RNA was extracted.
Growth protocol
U251 cells were maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum, amd incubated in 37 ºC in a humidified incubator with 5% CO2.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted usingTRIZOL Reagent (Cat#15596-018， Lifetechnologies, Carlsbad, CA, US)following the manufacturer’ s instructions and check ed for a RIN number to inspect RNA integration by an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, US).Qualified total RNA was further purified by RNeasy mini kit (Cat#74106, QIAGEN, GmBH, Germany) and RNase-Free DNase Set (Cat#79254, QIAGEN, GmBH, Germany).
Label
Cy3
Label protocol
Total RNA was amplified and labeled by Low Input Quick Amp Labeling Kit, One-Color (Cat#5190-2305, Agilent technologies, Santa Clara, CA, US), following the manufacturer’ s instructions. Labeled cRNA were purified by RNeasy mini kit (Cat#74106, QIAGEN, GmBH, Germany).
Hybridization protocol
Each Slide was hybridized with 1.65μg Cy3-labeled cRNA using Gene Expression Hybridization Kit (Cat#5188-5242, Agilent technologies, Santa Clara, CA, US) in Hybridization Oven (Cat#G2545A, Agilent technologies, Santa Clara, CA, US), according to the manufacturer’ s instructions. After 17 hours hybridization, slides were washed in staining dishes (Cat#121, Thermo Shandon, Waltham, MA, US) with Gene Expression Wash Buffer Kit(Cat#5188-5327, Agilent technologies, Santa Clara, CA, US), followed the manufacturer’ s instructions.
Scan protocol
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565CA) using one color scan setting for 4x44k array slides.
Description
Gene expression after 6h in control-infected U251
Data processing
Data were extracted with Feature Extraction software 10.7 (Agilent technologies, Santa Clara, CA, US). Raw data were normalized by Quantile algorithm, Gene Spring Software 11.0 (Agilent technologies, Santa Clara, CA, US).
