Snap fresh HCC tumor and adjacent non-tumor liver tissue was collected from resected HCC.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from frozen samples using miRNA isolation kits (Qiagen®, Germantown, MD) according to the manufacturer’s protocol. Briefly, around 30 mg of snap-fresh tissue of HCC or adjacent non-tumorous liver were disrupted and homogenized. The lysate was then centrifuged and the supernatant was transferred to the gDNA Eliminator spin column. After centrifugation, the flow-through was transferred to the RNeasy spin column. RNA was extracted using RPE and RW1. The gDNA Eliminator spin columns, gDNA Eliminator spin columns, and buffers were all supplied in the Qiagen miRNA isolation kits. The concentration and quality of total RNA were measured by NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE) at 260 and 280 nm (A260/280) and confirmed by gel electrophoresis.
Label
Cy3
Label protocol
Labeling was performed following Agilent's protocl. Briefly, 1 μg of total RNA was amplified by a low RNA input fluor linear amp kit (Agilent) and labeled with Cy3 (CyDye, PerkinElmer, Waltham, MA).
Hybridization protocol
Hybridization was performed following Agilent's protocl. Briefly, using incubation with fragmentation buffer at 60°C for 30 minutes, 1.65 μg of Cy3-labled cRNA was fragmented to an average size of about 50–100 nucleotides. Correspondingly fragmented labeled cRNA was then pooled and hybridized to ¬¬-SurePrint G3 ChIP/CH3 1X1M array (Agilent) at 60°C for 17 hours.
Scan protocol
Scanning was performed following Agilent protocol. Briefly, the microarrays were scanned with an Agilent microarray scanner at 535 nm for Cy3
Data processing
The data was processed using Agilent Feature Extraction, version 10.5, following Loess/Lowess algorithm and implementation. Per Gene normalization: normalize to median
