Human gene 2.0 st array

Total RNA was extracted using TRIzol (Invitrogen), and its quality was determined using a spectrophotometer (NanoDrop 2000) and an Agilent Bioanalyzer™ 2100 system. Thereafter, cRNA was synthesized from 10 ng RNA with the WT Pico Reagent Kit (Affymetrix) according to the manufacturer’s instructions. The samples were hybridized onto the Human Gene 2.0 ST Array (Affymetrix) for 17 h at 45 °C according to the manufacturer’s instructions. The arrays were scanned using a GeneChip Scanner 3000 7G (Affymetrix). After normalization, the genes related to differentiation and proliferation were selected and data processing was performed using GeneSpring GX13.1.1.

RNA from normal human cortex and white matter were obtained from Biochain, Origene, Clontech, and Agilent. Total RNA extraction, RT-qPCR protocol and ΔCt analysis were previously described [23 (link)]. Beta2 microglobulin or actin was used as endogenous control in the ΔCt analysis. Results of RT-qPCR are expressed either in 1/∆Ct values -used to express the levels of mRNA without comparison to a standard- or fold induction -used when we compared the levels of mRNA under a given condition to a standard.
After RT on GSCs RNA (at least three samples per cell line) with biotinylated desoxyribonucleotides, cDNA were hybridized on an Affymetrix Human Gene 2.0 ST array. Then, the DNA complexes were revealed by fluorescent streptavidin. Images were analyzed by Command Console and normalized with RMA method (data were normalized per cell line). Two lists of differentially expressed genes (CT1 vs PVZ1 and CT2 vs PVZ2) were established using a criteria based on adjusted p-value cut off of 0.001 and log2 fold change >0.65 or <-0.65. In the heatmap, we only showed the differentially expressed genes that were common to both lists and had the same directional change.

Total RNA from tumor and non-cancerous normal tissues obtained from three HCC patients was isolated using the TRIzol reagent (Invitrogen, Carlsbad, CA), amplified and transcribed into cDNA. The biotinylated cDNA was hybridized to the Human Gene 2.0 ST Array (Affymetrix, Santa Clara, CA), as performed by Chang Gung Memorial Hospital Genomics Center (Taiwan), following the manufacturer's instructions. The microarray data were analyzed using Transcriptome Analysis Console (TAC) software version 3.0 (Affymetrix).

The miRNA expression profile GSE84260 and the gene expression profile GSE73374 were obtained from the GEO database (http://www.ncbi.nlm.nih.gov/geo/). The GSE84260 dataset based on GPL15018 (Agilent Human miRNA V16.0 Microarray) contained 32 samples, including 16 PE placenta samples and 16 normal placenta samples. The GSE73374 dataset based on GPL16686 (Affymetrix Human Gene 2.0 ST Array) contained 36 samples, including 19 PE placenta samples and 17 normal placenta samples.

For gene expression array, 100 nanograms of total RNA from control and 200 μM SAM-treated MDA-MD-231 samples from three independent experiments was used. RNA quality and quantity were assessed using NanoDrop® ND-1000 spectrophotometer (Thermo Scientific, Wilmington, Delaware, USA) (260/280 >1.8 accepted) and Agilent 2100 Bioanalyzer (Waldbronn, Germany) (RIN 7 ≥ accepted). Gene expression profiling was performed using Affymetrix Human Gene 2.0 ST Array (Santa Clara, California, USA) at the Génome Québec Innovation Centre (McGill University) following standard protocols.
Data from the biological replicates were then normalized using the Robust Multi-array Average (RMA) method implemented in the Bioconductor package oligo [48 (link)]. Differential gene expression analysis was performed using the Bioconductor package Limma with a threshold defined by P <0.01 and |fold change| >1.5. The data was submitted to Gene Expression Omnibus (GEO) under the accession number of GSE98275.

Total RNA extracted from the dorsolateral prostate from two pairs of 24-week-old male C57BL/6 mice undergoing vasectomy or sham surgery at 10 weeks of age, using TRIzol (Invitrogen, Carlsbad, CA, USA), was subjected to microarray gene expression analysis at the University of Rochester Genomics Research Center, using Human Gene 2.0 ST Array (Affymetrix, Santa Clara, CA, USA). Scanned fluorescence signals were converted to continuous values by the Gene Expression Console software (Affymetrix).