cell line: Hepatocellular carcinoma cell line MHCC-97H genotype/variation: UGT2B15 deficiency
Treatment protocol
transfection with shRNA for 7 days
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 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
SH2_NS Perform human gene expression microarray after transfection with shRNA for 7 days
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. Fold Change (linear) =< 0.5 or Fold Change (linear) >= 2, T-test p-value < 0.05
