tissue: lung adenocarcinoma ifn-gamma level: high pd-l1 level: high tumor stage: IIIA gender: male age: 52y
Treatment protocol
Lung adenocarcinoma specimens were obtained from patients, who underwent pulmonary resection for lung cancer between 2012 and 2015 in the Department of Thoracic Surgery, Tongji Hospital. Each tumor sample was divided into two parts. One part of the tumor tissue was stored in liquid nitrogen for RNA and Protein extraction. The other part was fixed in 10% formalin and embedded in paraffin wax for immunohistochemistry. None of these patients received either radiation or chemotherapy before surgical operation. Histological diagnosis of tumors was based on the World Health Organization criteria. The TNM stage was determined according to the revised 2010 AJCC staging guidelines.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using TAKARA RNAiso Plus#9109 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 mini kit (Cat#74106, QIAGEN, GmBH, Germany) and RNase-Free DNase Set (Cat#79254, QIAGEN, GmBH, Germany).
Label
Cy3
Label protocol
Total RNA was amplified and labeled by Low Input Quick Amp Labeling Kit, One-Color (Cat.# 5190-2305, Agilent technologies, Santa Clara, CA, US), following the manufacturer’s instructions. Labeled cRNA were purified by RNeasy mini kit (Cat.# 74106, QIAGEN, GmBH, Germany).
Hybridization protocol
Each slide was hybridized with 600 ng Cy3-labeled cRNA using Gene Expression Hybridization Kit (Cat.# 5188-5242, Agilent technologies, Santa Clara, CA, US) in Hybridization Oven (Cat.# G2545A, Agilent technologies, Santa Clara, CA, US), according to the manufacturer’s instructions. After 17 hours 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), followed the manufacturer’s instructions。
Scan protocol
Slides were scanned by Agilent Microarray Scanner (Cat#G2565CA, Agilent technologies, Santa Clara, CA, US) with default settings, Dye channel: Green, Scan resolution=3μm, PMT 100%, 20bit.
Description
gene expression in IIIA lung adenocarcinoma with IFN-γ high expression and PD-L1 high expression
Data processing
Data were extracted with Feature Extraction software 10.7 (Agilent technologies, Santa Clara, CA, US). Raw data were normalized by Quantile algorithm, limma packages in R.
