Total RNA was extracted and purified using RNeasy micro kit and checked for a RIN number to inspect RNA integration by an Agilent Bioanalyzer 2100.
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.
Hybridization protocol
Each slide was hybridized with 1.65μg 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. Data were extracted with Feature Extraction software 10.7 (Agilent Technologies, Santa Clara, CA, US).
Description
Control 3 2
Data processing
Raw data were normalized by Quantile algorithm, limma packages in R. After the original data were normalized with the limma package in the software R, the differential genes were screened using Fold-change (fold of expression difference) and T-test (Student's t-test) statistical methods, and the selection conditions were as follows: 1) Fold Change (linear) =< 0.5 or Fold Change (linear) >= 2. 2) T-test p-value <0.05 or <0.01.
