PBMCs were isolated using 1.078 Optiprep （Axis-shield, Oslo, Norway) density gradient according to Application Sheet C08. Monocytes were allowed to adhere to plastic cell flasks for 24 hours and derived to macrophages after 5 days. Macrophages were challenged by M. bovis at MOI of 10 and unattached bacteria are washed out by PBS after 2 hours. At 6 hour, TRIzol (Invitrogen, USA) was added to each sample.
Growth protocol
Cows were recruited from a farm in Shandong province in China and were performed single intradermal tuberculin test and the INF-gamma release test (Bovigam, Prionics AG).
Extracted molecule
total RNA
Extraction protocol
TRIzol treated samples were purified by PureLink RNA Mini Kit (Ambion, USA). The quality and quantity of total RNA was examined by NanoDrop and Agilent Bioanalyzer 2100 (Agilent technologies, USA). Total RNA was further purified by Rneasy micro kit (QIAGEN, 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), followed the manufacturer’s instructions. Labeled cRNA were purified by RNeasy mini kit (Cat#74106, QIAGEN, 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 manufacture's instructions. After 17 hours hybridization, slides were washed in staining dishes (Cat#121, Thermo Shandon, MA, USA) with Gene Expression Wash Buffer Kit (Cat#5188-5327, Agilent technologies, US), followed the manufacturer's instructions.
Scan protocol
Slides were scanned by Agilent Microarray Scanner (Cat#G2565CA, Agilent technologies, US) with default settings, Dye channel: Green ,Scan resolution=5μm, PMT 100%, 10% 16bit.
Description
1- Gene expression at 6 hour
Data processing
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).
