All the specimens were fixed in 10% buffered formalin and embedded in paraffin.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted and purified using RecoverAllTM Total Nucleic Acid Isolation (Cat # AM1975, Ambion, Austin, TX, US) |, following the manufacturer’s instructions and checked for a RIN number to inspect RNA integration by an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, US).
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 immediately after washing on the Agilent DNA Microarray Scanner (Cat # G2565CA，Agilent technologies, Santa Clara, CA, US) using one color scan setting (Scan resolution 3um, and Green PMT is set to 100%).
Description
miRNA expression in primary tumor of case1
Data processing
The scanned images were analyzed with Feature Extraction Software 10.7.1.1 (Agilent) with default settings. Raw data were normalized by Quantile algorithm using Gene Spring Software 12.6 (Agilent technologies, Santa Clara, CA, US). Raw data were normalized by Quantile algorithm.
