Agilent scanner g2565ba

Frozen kidney tissues were first placed on the RNAlater^®-ICE (Life technologies) for 16 h at −20 °C before the subsequent homogenization process. Total RNA was isolated using the miRNeasy kit (Qiagen) following the manufacturer’s instructions and was quantified with a Nanodrop spectrophotometer (ND-1000, Nano drop Technologies, DE, USA). The input for the Agilent miR labeling system was 100 ng total RNA. Dephosphorylated and denatured total RNA was labeled with cyanine 3-pCp and subsequently hybridized to the Agilent mouse miR microarray release version 15 using the miR Complete Labeling and Hyb Kit (Agilent Technologies, Inc., Santa Clara, CA, USA). Following hybridization for 20 h, the slides were washed with a Gene Expression Wash Buffer Kit (Agilent) and measured using an Agilent Scanner G2565BA. Agilent Feature Extraction Software (version 9.5.1) and GeneSpring GX software (version 12.5, Agilent) were used for data processing, analysis, and monitoring.

After addition of spike-in controls, 200 ng of RNA was amplified and labeled using one-color microarray based gene expression protocol v 6.0; labeled cRNAs were hybridized to Human GE 4×44K v2 Microarray (Agilent Technologies, Santa Clara, CA). Slides were scanned by the Agilent G2565BA scanner and images were processed by Feature Extraction software v.10 (Agilent Technologies). The raw data were imported into GeneSpring 12.1 software (Agilent Technologies) and a quantile normalization was applied.
Microarray data have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus and are accessible via: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=xxsxlqouuaasmzk&acc=GSE44099.

Three sample RNA pools were run per sub-array with three individual RNA pool sub-arrays per condition. Agilent microarray was performed (Agilent 5190-2305, 5188-5282, 5188-5242, 5188-5327, and RNeasy mini kit, Qiagen 74104) following the labeling protocol (version 6.6, September 2012 Agilent Technologies, G4140-90040) per manufacturer’s instructions and scanned using the Agilent G2565BA scanner.
Data analysis was performed using the Bioconductor package Limma in the programing language “R.” Target files were created containing Agilent G2565BA microarray scanner output files. Background correction, cyclic loess normalization, and averaging of repeats was performed. A linear modeling matrix was built and fitted. Gene lists were filtered discarding those unaltered by IL-1ß in the order of a log2 fold-change of < 1 and for an adjusted P value of < 0.05. A pathway analysis functional output was obtained using Signaling Pathway Impact Analysis (SPIA) in R. All was as described in previous papers from our group.^{13 (link)} A two-dimensional projection of the microarray expression data was generated using the non-parametric dimensionality reduction. This was achieved using the t-distributed stochastic neighbor embedding (t-SNE) algorithm in the R package Rtse. The resulting t-SNE output was plotted with R package ggplot2. The array data will be deposited in NCBI’s Gene Expression Omnibus.