Agilent microarray scanner

RNA was reversed transcribed into cDNA and then labeling. Chip hybridization was performed using an Affymetrix (Thermo Fisher Scientific, Inc.) expression profile chip and GeneChip Hybridization Wash and Stain kit. The results were scanned with an Agilent microarray scanner and read with the Feature Extraction software 10.7.

In this study, array-based comparative genomic hybridization (aCGH) method was used in order to detect deletions and duplications in tissue samples. Genomic DNA was isolated from tissue samples by using DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany). DNA quality was determined by agarose gel electrophoresis and quantity was assessed by spectrophotometer (NanoDrop ND-1000; NanoDrop Technologies, Wilmington, DE). CytoChip Focus Constitutional (BlueGnome, Cambridge, UK) was used as aCGH platform. Patient DNA with a sufficient quality and quantity and reference DNA (Human Genomic DNA: Female; Promega Corporation, Madison, USA) were labelled according to CytoChip protocol. Labelled patient DNA and reference DNA were combined and they were left for hybridization at 47 °C for 20 hours with aCGH microchips according to the protocol. Microchips that were rinsed by using various dilutions of 20XSSC following hybridization period according to the protocol were scanned by Agilent Microarray Scanner (Agilent Microarray Scanner; Agilent Technologies, Palo Alto, CA). Scanned images were numerically examined and all chromosomal copy number ratios were analyzed for deletions and duplications by using fixed CytoChip algorithm settings in BlueFuse Multi v2.2 (BlueGnome) software program.

Agilent oligonucleotide technology was applied to profile the differentially expressed miRNAs between normoxia and hypoxia (8 h) groups. In brief, the RNA was isolated by mirVanaTM miRNA Isolation Kit (Cat#AM1560, Ambion, Austin, TX, US) and followed by labeling and hybridization with miRNA Complete Labeling and Hyb Kit (Cat#5190-0456, Agilent technologies, Santa Clara, CA, US). The microarray used for hybridization was Agilent human miRNA (8*60 K) V18.0. Then slides were scanned by Agilent Microarray Scanner (Cat#G2565BA, Agilent technologies, Santa Clara, CA, US) and Feature Extraction software 10.7 (Agilent technologies, Santa Clara, CA, US) with default settings. Raw data were normalized by Quantile algorithm, Gene Spring Software 11.0.
The microarray data was submitted to GEO database and the GEO accession number was GSE68593.

Total RNA of the cells was extracted by using a TRIzol commercial kit (Invitrogen, USA). And Quick Amp Labeling Kit, One-Color (Agilent, USA) was used to prepare labeling reaction. Then labeled/amplified RNA and labeled cRNA QC were purified by RNeasy Mini Kit (Qiagen, German). After fragmentation, hybridization, and microarray wash, the microarray was scanned by Agilent Microarray Scanner (Agilent, USA). LncRNAs with differential expressions in primary microglial cells were picked out by the whole genome microarray expression profiling with the fold change > 2 and adjusted P < 0.05. The microarray analysis was performed with Agilent Feature Extraction by Oebiotech, Shanghai, China. In addition, sample preparation and microarray hybridization were performed according to the manufacturer’s standard protocol with only minor modifications.

Slides were scanned by Agilent Microarray Scanner (Cat#G2565CA, Agilent technologies, Santa Clara, CA, US) with default settings, Dye channel: Green, Scan resolution = 3 μm, PMT 100%, 20bit. 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. We slect a standard threshold set for differentially expressed genes of a fold change≥2.0 and a 푝 value≤0.05.