Gene chip expression analysis
Gene chip expression analysis was performed by Kangcheng Biotechnology Co., Ltd. (Shanghai, China) using the Agilent array platform. First, the RNA quantity and quality were assessed using a NanoDrop ND-1000 UV Spectrophotometer (Implen, German). RNA integrity of each sample was identified by standard denaturing agarose gel electrophoresis. Second, sample labeling and chip hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent, California, USA). The samples were labeled by using the Agilent Quick Amp Labeling kit and hybridization experiments were performed by using the Agilent SureHyb. Specifically, the total RNA of each sample was linearly amplified and labeled with Cy3-UTP; the labeled cRNAs were purified by using the RNeasy Mini Kit (Qiagen, Dusseldorf, German), and the concentration and activity were detected with a NanoDrop ND-1000 UV spectrophotometer. The purified Cy3-UTP-labeled cRNAs were hybridized with Agilent mouse 4×44K gene expression profiling chip v2 (Agilent, California, USA). The hybridization chip was washed, fixed, and scanned by using Agilent DNA Microarray Scanner (part number G2505C); Chip probe signal values were acquired by using Agilent Feature Extraction software (v11.0.1.1) to obtain raw data. Finally, the Quantile normalization of raw data and subsequent data processing were performed by using GeneSpring GX v12.1 software (Agilent, California, USA).