Luxscan 10k microarray scanner

We used a non-commercial microarray assay as previously reported^{11 (link),45 (link),50 (link),51 (link)}. Briefly, the probes (40 μM final concentration) mixed with printing buffer were printed onto slides in duplicate using SmartArrayer™ 136 printer (CapitalBio Inc, Beijing, China); RNA extracted from 600 μl serum was dephosphorylated and labeled with 100 nmol l⁻¹ 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. The labeled RNAs were mixed and hybridized to the array with a 2× hybridization solution (final concentration: 5× Denhart’s solution, 0.5% sodium dodecyl sulfate (SDS), 5× SSC) in a Hybridization Chamber (Corning Inc, Corning, NY, USA) at 46 °C for 12–16 h. After washing, slides were scanned with a LuxScan 10 K Microarray Scanner (Capital Bio, Beijing, China), and images were analyzed with th GenePix Pro 6.0 software (Axon Instruments, Foster City, CA, USA). Expression data were normalized through quantile normalization and the RMA algorithm. Then miRNAs with significantly different expression between sensitive and resistant patients were selected. The microarray data have been deposited in the Gene Expression Omnibus database (GSE101841).

Tet-Off-inducible RBM24 stable cells were cultured with doxycycline. For miRNA array analysis, doxycycline was removed from the culture medium after 48 h, and total RNA was isolated as described previously.^{52 (link)} Next, a microarray containing 873 miRNA probes was employed according to previously described miRNA probe design, RNA labeling and microarray hybridization methods.^{53 (link), 54 (link)} Briefly, 2.5 μg total RNA was labeled with pCp-DY647 (Dharmacon, Lafayette, CO, USA). After hybridization, the arrays were scanned with a LuxScan 10 K Microarray Scanner (CapitalBio, Beijing, China), and the resulting images were analyzed with GenePix Pro 6.0 software (Axon Instruments, Foster City, CA, USA).

The paraffin-embedded tissues from the 63 patients with ICC were obtained from the Department of Pathology, Sun Yat-Sen University Cancer Center. We cut five sections with 10 μm thickness from each patient, and mounted them onto glass slides. Tumor areas (containing > 90% tumor tissue) were scraped off with a scalpel under a microscope and collected in nuclease-free microcentrifuge tubes. The NIBDs were peeled off from the resected liver. Total RNA was extracted from ICC and NIBD with an acid phenol-chloroform extraction method, followed by ethanol precipitation, as described previously [27 (link)]. Quantity and quality of RNA were measured by using a NanoDrop™ 1000 (Thermo Fisher Scientific, MA, USA) spectrophotometer.
Total RNA (2.5 μg) from each sample was used for labeling with pCp-DY647 (Dharmacon, Lafayette, CO, USA) and hybridized in accordance with published protocols [15 (link)]. After hybridization, the microarray was scanned with a LuxScan 10 K Microarray Scanner (CapitalBio, Beijing, China) and the scanned images were gridded by using GenePix Pro 6.0 software (Axon Instruments, Foster City, CA, USA).