Tissue sections were cut from frozen blocks and then stained with cresyl violet for total RNA (Ambion, Austin, TX, USA). Carcinoma cells or non-malignant cells were collected from cancerous or adjacent noncancerous tissue sections using LMD (Leica Microsystems, Wetzlar, Germany) by pathologists
Growth protocol
A total of 3 SCEC tissues and matched adjacent noncancerous tissues were dissected from the surgical specimens and snap-frozen in liquid nitrogen, then stored at -80°C in the tissue bank of Fudan University Shanghai Cancer Center
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using TRIZOL Reagent (Cat#15596-018，Life technologies, Carlsbad, CA, US)following the manufacturer’s instructions and checked for a RIN number to inspect RNA integrity by an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, US).Qualified total RNA was further purified by RNeasy micro kit (Cat#74004, QIAGEN, GmBH, Germany) and RNase-Free DNase Set (Cat#79254, QIAGEN, GmBH, Germany).
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 6 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
Hybridization protocol
Array hybridization and wash was performed using GeneChip Hybridization, Wash and Stain Kit (Cat#900720, Affymetrix, Santa Clara, CA, US)in Hybridization Oven 645 (Cat#00-0331-220V, Affymetrix, Santa Clara, CA, US)and Fluidics Station 450 (Cat#00-0079, Affymetrix, Santa Clara, CA, US) followed the manufacturer’s instructions.
Scan protocol
The gene chips were scanned using a confocal laser scanner (GeneArray Scanner 3000). The scanned image was converted into numerical values of the signal intensity by Command Console Software 3.1 (Affymetrix) with default settings.
Description
gene expression data from SCEC patient2
Data processing
Raw data were subjected to Robust Multiarray Average quantile normalization analysis (RMA) algorithm by GeneSpring GX 12.5 Software (Agilent technology, Santa Clara, CA, US) to generate .CEL intensity files
