TAMs were isolated from tumor tissue and total RNA was extracted from cells using Trizol (Invitrogen, CA, USA) reagent.Total RNA from each sample was quantified by the NanoDrop ND-1000 and RNA integrity was assessed by standard denaturing agarose gel electrophoresis. RNA quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
Label
Cy3
Label protocol
miRNA molecular in total RNA was labeled by miRNA Complete Labeling and Hyb Kit(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(Agilent technologies, Santa Clara, CA, US) in hybridization Oven(Agilent technologies, Santa Clara, CA, US)at 55℃，20rpm for 20 hours according to the manufacturer’s instructions, hybridization ection. After hybridization, slides were washed in staining dishes (Thermo handon, altham, MA, US) with Gene Expression Wash Buffer Kit（Agilent technologies, Santa Clara, CA, US).
Scan protocol
Slides were scanned immediately after washing on Agilent Microarray Scanner(G2565BA, Agilent technologies, Santa Clara, CA, US） Feature Extraction software 10.7 (Agilent technologies, Santa Clara,CA, US)with default settings.
Description
Sample name: fuM_NS expression of microRNA
Data processing
The scanned images were analyzed with Feature Extraction software 10.7 (Agilent technologies, Santa Clara, CA, US)with default settings. Raw data were normalized by Quantile algorithm, Gene Spring Software 11.0(Agilent technologies, Santa Clara, CA, US).
