mRNA Microarray Experiment

The samples used for mRNA microarray were the same as the miRNA microarray. Microarray was performed by a service provider (CapitalBio, China). The microarray (Probe length 60-mer, 135K Format) containing 44987 probe sets was provided by Roche-NimbleGen. For each sample, 1 µg of total RNA was treated with the CapitalBio cRNA Amplification and Labeling Kit (CapitalBio, China) according to the manufacturer’s instructions. After reverse transcribed with a T7 oligo(dT) primer, second-strand synthesis and purification, the generated cDNAs were in vitro transcribed to synthesize multiple copies of cRNAs. Then 5 µg of purified cRNAs were reverse transcribed with random primer. Labeled cDNA molecules were generated by subsequent Klenow Fragment Polymerase labeling with Cy3-dCTP (GE Healthcare, USA). Following purification and drying, the labeled cDNAs were then dissolved in 80 µl hybridization buffer (3×SSC, 0.2%SDS, 5×Denhart’s, 25% formamide). Hybridizations were performed overnight at 42°C using hybridization system 12 (Roche NimbleGen, USA). The arrays were then washed and dried. The fluorescence intensity was collected using NimbleGen MS 200 Microarray Scanner. Data were extracted from scanned images using NimbleScan v2.6 software. Quantile normalization RMA (Robust Multi-Array) analysis was performed to generate gene expression values. The differences between the two groups were analyzed using SAM (Significance Analysis of Microarrays) method with SAMR software version 3.02 [42]. The FDR (False Discovery Rate) were calculated. Differentially expressed genes (DEGs) were selected with FDR <5% and FDR <10%. All data were MIAME compliant and have been deposited in GEO (accession number GSE33524).