Total RNA was extracted using an acid phenol/chloroform extraction method followed by ethanol precipitation.
Label
Cy3-dUTP
Label protocol
2.0 μg of total RNA of intrahepatic cholangiocarcinoma/normal intrahepatic bile duct tissue was employed with 100 nmol/L of pCp-DY647 (Dharmacon, Lafayette, CO, USA) and 15 Units of T4 RNA ligase (USB, Cleveland, Oh, USA) in a total reaction volume of 20 µL at 16°C overnight
Hybridization protocol
The labeled RNAs were hybridized to the array with a 1×hybridization solution (5× Denhart’s solution, 0.5% SDS, 5× SSC) in a Hybridization Chamber (Corning Inc, Corning, NY, USA) at 46°C for 12 to 16 hours.
Scan protocol
Scanned in a LuxScanTM -10K Scanner (capitalBio Inc.,Beijing, China).
Data processing
Quantile normalization across multiple arrays, log 2 transformation of expression levels,EXPression Analyzer and DisplayER (EXPANDER) version 4.1 was used.
