Genechip wt terminal labeling and hybridization kit

Total RNA concentrations from brain samples were measured with a Nanodrop 1000 Spectrophotometer (ThermoFisher) and RNA integrity was assessed using the Agilent 2100 BioAnalyzer (Agilent Technologies, USA). Gene expression patterns were analyzed using Genechip© Mouse Clariom S 24 × arrays plates (Affymetrix, ThermoFisher). Starting material was 100 ng of total RNA of each sample. Briefly, sense ssDNA was generated from total RNA with the GeneChip WT Plus Reagent Kit (Affymetrix) following the manufacturer’s instructions. Then, sense ssDNA was fragmented, labeled and hybridized to the arrays with the GeneChip WT Terminal Labeling and Hybridization Kit (Affymetrix). Arrays plates were scanned and processed with Affymetrix GeneChip Command Console to obtain expression array intensity.cel files.

Total RNA was extracted from 1x10⁷ cells induced for 24 h with 1 μM ß-estradiol using the Qiagen RNeasy Mini Kit. Expression analysis starting from 100 ng of total cellular RNA was performed using the Ambion WT Expression Kit (Applied Biosystems) and subsequently the GeneChip WT Terminal Labeling and Hybridization Kit (Affymetrix) followed by the GeneChip Human Gene 2.0 ST array (Affymetrix) according to the manufacturer's protocol. All affymetrix CEL files have been processed in Bioconductor/R using robust multiarray average (RMA) for normalization and summarization and limma for differential expression and significance. Quality has been checked using the array QualityMetrics package. Additional filtering based on the fold change between the two conditions was applied with different stringency, individually described in the legend of the tables and figures. Analyzation and visualization of the Microarray was performed using Genesis, available at http://genome.tugraz.at. Quantitative RT-PCR analysis was performed as described previously [72 (link)]. Primers used for RT-qPCR were designed applying Primer3 software (http://primer3.ut.ee/) and selection of mature transcripts was ensured by amplification across exon-exon junctions. Primers used for quantitative RT-PCR are summarized in S3 Table. All data were normalized for the relative abundance of the Actin B transcript.

Gene expression profiling analysis in retinas of 8-week old diabetic (db/db) mice and non-diabetic (db/+) controls was performed using Mouse Gene 1.0 ST DNA arrays (Affymetrix, UK). For this purpose, total RNA was first isolated from retinas (n = 4/group) using the RNeasy Mini Kit (Qiagen, Germany) and 200 ng were then used to synthesize sense ssDNA with the Ambion WT Expression Kit (Life Technologies, UK). Next, ssDNA was fragmented, labeled and hybridized onto DNA microarrays using the GeneChip WT Terminal Labeling and Hybridization Kit and the GeneTitan platform (Affymetrix, UK), following the manufacturer’s instructions. Microarray Analysis Suite 5.0 software was used to process the microarray images and analysis of the data obtained was performed by the Statistics and Bioinformatics Unit of the Vall d’Hebron-Research Institute using the open source software Bioconductor.

The microarray experiment was conducted according to previously published procedures [19 (link)–22 (link)]. The protocol involving preparation of RNA for further hybridization was carried out using GeneChip WT PLUS Reagent Kit (Affymetrix, Santa Clara, CA, USA). The first stage included performing a two-step cDNA synthesis reaction from 100 ng RNA with the use of random primers extended by the T7 TNA polymerase promoter. The cRNA was synthesized during in vitro transcription (16h, 40°C) and then purified, re-transcribed into cDNA, and biotin labeled and fragmented using the Affymetrix GeneChip WT Terminal Labeling and Hybridization kit (Affymetrix, Santa Clara, CA, USA). Biotin-labeled fragments of cDNA were hybridized to the microarray probes included in the Affymetrix Human Gene 2.1 ST ArrayStrip (20h, 48°C). The microarrays were then washed and stained using the Affymetrix GeneAtlas Fluidics Station (Affymetrix, Santa Clara, CA, USA). The Imaging Station of GeneAtlas System (ThermoFisher Scientific, MA, USA) was used to scan the array strips. Preliminary analysis of the scanned chips was conducted with the Affymetrix GeneAtlas Operating Software (Affymetrix, Santa Clara, CA, USA). The software’s quality criteria were applied to verify the quality of the gene expression data.