cell line: hepatoma cell line SMMC-7721 genotype/variation: YWHAZ knockdown
Treatment protocol
Each well of SMMC-7721 cells in six well plates was transfected by 10 nmol si-YWHAZ or the control siRNA-NC using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s instructions.
Growth protocol
SMMC-7721 cells were cultured in six well plates and incubated in a 37 °C, 5% CO2 humidified incubator
Extracted molecule
total RNA
Extraction protocol
48h after transient introduction of si-YWHAZ or siRNA-NC, total RNA was extracted and 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 after silencing YWHAZ
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.
