Beadstudio application

PLC-PRF-5 cells transfected with control plasmids, pCDNA3.1–Twist1, or M02–TP; added with thymidine (dT); or added with dT after transfection of M02-TP were collected with TRIzol (Sigma, USA) for RNA isolation. After preparation of biotinylated complementary RNA (cRNA) by using the Illumina TotalPrep RNA Amplification Kit (Ambion), cRNA was hybridized with BeadChip. The samples were prepared as technical duplicates. Data were acquired with Illumina BeadChip Reader and evaluated using Illumina BeadStudio Application by Genergy Bio (China). Gene set enrichment analysis (GSEA)^{21 (link)} and gene ontology analysis were used to detect changes in biological processes, cellular components, and molecular functions. Pathway analysis was conducted based on the KEGG database. Protein–protein interaction (PPI) was analyzed using the STRING database^{22 (link)} and Cytoscape software (NRNB)^{23 (link)}.

RNA was converted to labeled cRNA and hybridized to Illumina mouse WG-6_v2_Bead Chip. Microarray Hybridization, washing and detection were performed using the Illumina Gene Expression System K it according to the manufacturer's protocol. Arrays were scanned with an Illumina BeadArray Reader confocal scanner. Initial microarray gene expression data were obtained using the gene expression analysis module of Illumina BeadStudio Application.

An Illumina HumanHT-12 v4 Expression BeadChip (Illumina, San Diego, CA, USA) analysis containing 47,231 probes was processed by Macrogen (Seoul, Korea). Briefly, biotinylated cDNA was prepared from total RNA using the Illumina RNA amplification kit (Ambion, Austin, TX, USA). Fluorescent signals were obtained by an Illumina BeadArray Reader, and data extraction and analysis were performed with an Illumina BeadStudio Application. Data were processed by excluding genes with P-values over 0.05, and differentially expressed genes were obtained displaying at least a 1.5-fold difference between the control cells and the tested cells. The array data were uploaded to the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/) with accession numbers GSE112518 and GSE112517 for overexpression and knock-down data of KLHDC7B and GSE112519 for knock-down data of STAR1. Significantly changed gene pools obtained from expression array were further analyzed with Ingenuity Pathway Analysis (IPA) (Qiagen; www.qiagen.com/ingenuity) to construct disease and function annotation, and network map. Each disease and function were listed based on the hierarchical order of the activation Z-score. The top network was nominated with the highest confidence among the networks.