RNA was prepared using the QIAamp Circulating Nucleic Acid Kit (Qiagen) following the manufacturer's recommendations. RNA was quantified using a NanoDrop-1000 spectrophotometer and 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 (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，20 rpm 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 11.5 (Agilent technologies, Santa Clara, CA, US) with default settings.
Description
Cerebrospinal fluid before initial leptomeningeal metastasis-directed therapy
Data processing
The scanned images were analyzed with Feature Extraction Software 11.5 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
