SH-SY5Y and U87MG cells were seeded at a density of 3×105 cells/well in 12-well plate overnight to adhere. Cells were washed twice with phosphate buffer saline (PBS) and infected with HSV-1 strains of 17syn+、Kos and Mckrae at an MOI of 0.01, respectively.
Growth protocol
Human neuroblastoma cells SH-SY5Y and human glioblastoma cells U87MG were purchased from American Type Culture Collection (ATCC, VA, USA) and stored in Department of Viral Immunology, Institute of Medical Biology, Chinese Academy of Medical Sciences (Kunming, China). The above tow types of cells were cultured in Dulbecco′s modified Eagle′s medium (DMEM, Thermo Fisher Scientific, MA, USA), supplied with 10% (V/V) fetal bovine serum (FBS, Gibco, MA, USA) and penicillin–streptomycin (100 U/mL and 100 μg/mL). The cells were maintained at 37C in a humidified atmosphere containing 5% CO2.
Extracted molecule
total RNA
Extraction protocol
The total RNA of infected cells was extracted and purified using miRNeasy Mini Kit (QIAGEN, GmbH, Germany) according to the manufacturer’s instructions at the time point of 0, 4 and 10 hpi. The RNA concentration was measured by NanoDrop ND-2000 spectrophotometer (Thermo, MA, USA) and RNA integrity number (RIN) was determined by 2100 Bioanalyzer (Agilent technologies, CA, USA) and agarose gel electrophoresis.
Label
Cy3
Label protocol
miRNA molecular in total RNA was labeled by miRNA Complete Labeling and Hyb Kit(Cat #5190-0456, Agilent technologies, Santa Clara, CA, US)followed the manufacturer’s instructions, labeling section.
Hybridization protocol
Each slide was hybridized with 100ng Cy3-labeled RNA using miRNA Complete Labeling and Hyb Kit (Cat # 5190-0456, Agilent technologies, Santa Clara, CA, US) in hybridization Oven (Cat # G2545A, Agilent technologies, Santa Clara, CA, US) at 55°C，20rpm for 20 hours according to the manufacturer’s instructions, hybridization section. After 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).
Scan protocol
Slides were scanned by Agilent Microarray Scanner (Cat # G2565CA, Agilent technologies, Santa Clara, CA, US) and Feature Extraction software 10.7 (Agilent technologies, Santa Clara, CA, US) with default settings.
Description
miRNAs expression profile infected by HSV-1 in SH-SY5Y cells
HSV-1 infection of neural system-derived cells, including SH-SY5Y and U87MG cells at 0, 4 and 10 hour post infection with an MOI of 0.01,to identify the differentially expressed miRNAs and perform pathway enrichment analysis using KEGG.
