Genechip human genome u133 plus 2.0 microarray

Total RNA was extracted from tissues and cells using a mirVana™ miRNA Isolation Kit (Ambion, Carlsbad, CA, USA) according to the manufacturer’s protocols. RNA samples OD260/OD280 ratios ranging from 1.9–2.0 were considered good quality. ABI® Reverse Transcription Kit (Applied Biosystems, Foster City, CA) was used for reverse transcription. Quantitative polymerase chain reaction (PCR) measurements for miR-let-7b-5p and miR-let-7c-5p were performed using TaqMan MicroRNA Assays (Applied Biosystems, Foster City, CA). Relative expressions of miR-let-7b-5p and miR-let-7c-5p were calculated using a comparative CT method with an internal control RNU6B for miRNA. The mRNA expression of target genes was detected by microarray hybridization and gene expression analyses that were performed using the GeneChip Human Genome U133 Plus 2.0 microarrays (Affymetrix) and Affymetrix GeneChip System (Biotechnology, Shanghai, China).

Affymetrix GeneChip™ Human Genome U133 Plus 2.0 microarrays were used for mRNA profiling. Microarray analysis was performed at Mayo Microarray Core facilities by technologists following standard procedures. Briefly, RNA samples were subject to Agilent analysis for quality controls. cDNA was prepared from 10 μg of RNA, quantified by spectrometry, and used as a template for the synthesis of biotinylated cRNA using RNA transcript labeling reagent (Affymetrix, Santa Clara, CA, USA). The quality of the cRNA probes was verified by gel electrophoresis and pilot hybridization with the Test-3 array. Hybridization solution containing fragmented cRNA probes and control cRNA (BioB, BioC, and BioD) was supplemented with herring sperm DNA and bovine serum albumin. The probe solution was heated at 99 °C for 5 min followed by incubation at 45 °C for 5 min before use. Hybridization was carried out at 45 °C for 16 h with constant rotation at 60 rpm. The arrays were washed and stained with streptavidin-phycoerythrin (Molecular Probes, Eugene, OR, USA). After washes, arrays were scanned using the GeneChip system confocal scanner (Hewlett Packard, Palo Alto, CA, USA).

Total RNA was purified from the cell lysates of hiPSCs, OVs, RGCs and RPE cells using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. RNA was analyzed using GeneChip Human Genome U133 Plus 2.0 microarrays (Affymetrix) according to the manufacturer’s instructions. Two biological replicates were analyzed for each cell type. The microarray data analysis was performed in the R (v.3.2.2) environment using Bioconductor packages (http://www.bioconductor.org). The raw intensity data were background corrected, log₂ transformed and quantile normalized using the Robust Multi-array Average (RMA) algorithm implemented by the affy package [28 (link)]. The differentially expressed genes (DEGs) were further identified using the limma package [29 (link)]. The probe sets that demonstrated Benjamini–Hochberg adjusted p < 0.01 and log₂ fold-changes of more than 1 or less than −1 (OV vs hiPSC, RGC vs hiPSC and RPE vs hiPSC) were selected for further analysis as DEGs. The microarray data were deposited in the NCBI Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/) and are accessible via the GEO accession number GSE96853. PANTHER software (version 11.1) was used for statistical overrepresentation analysis (binomial test) of GO-Slim terms, PANTHER Pathways and PANTHER Protein Classes in the lists of DEGs [30 (link)].

Total RNA and gDNA were purified from randomized lysates with the AllPrep kit (Qiagen Valencia, CA). cDNA synthesis, amplification and labeling were performed with the Ovation Pico WTA System vV2 and Encore Biotin (NuGEN Technologies, San Carlos, CA) with 2 ng of total RNA. Labeled hybridization targets were hybridized to GeneChip HumanGenome-U133 Plus 2.0 microarrays (Affymetrix, Santa Clara, CA). Nucleic acid extractions and microarray assays were performed at Oregon Health & Science University (OHSU Gene Profiling Shared Resource).

Laser microdissection was performed to procure the epithelial components of 20 ovarian tumor samples for RNA extraction as described previously [28 (link)]. During dissection, areas of interest in the sections were carefully outlined. Areas with immune cell and blood vessel infiltration were excluded to minimize contamination. Purified RNA samples were amplified, labeled, and hybridized onto GeneChip Human Genome U133 Plus 2.0 microarrays (Affymetrix, Santa Clara, CA) according to the manufacturer’s protocol. After hybridization, arrays were washed and stained using an Affymetrix Fluidics Station 450 and then scanned using a GeneChip Scanner 3000 7G. Microarray data were deposited into the National Center for Biotechnology Information Gene Expression Omnibus database with the accession number GSE54388.