Agilent microarray scanner g2565ba

Total RNA was isolated by Trizol, and RNA quality was checked using denaturing agarose gels. For microarray, the cRNA was purified and then hybridized to the 4×44K oligo microarray manufactured by Agilent Technologies. Microarray data were analyzed using an Agilent G2565BA Microarray Scanner. Comparative analysis was done using GeneSpring GX 10 (Agilent Technologies) [2 (link), 25 (link)–26 (link)]. The experimental data were compared to baseline data. Genes were considered differentially expressed if expression changed by at least two-fold.

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.

The microarray experiment included two biological replicates per treatment. All sample-labelling, hybridization, washing, and scanning steps were conducted according to the manufacturer’s specifications. Briefly, Cy3-labelled cRNA was generated from 50 ng input total RNA using the One Color Quick Amp Labelling Kit (Agilent Technologies Inc.). For every sample, 600 ng cRNA from each labelling reaction (with a specific activity above 9.0) was hybridized using the Gene Expression Hybridization Kit (Agilent Technologies Inc.) to the Agilent Whole Human Genome Oligo Microarray (Agilent Technologies Inc.), which is in a 4 × 44k 60-mer slide format, where each of the 4 arrays represents approximately 41,000 unique genes and transcripts. After hybridization, the slides were washed and then scanned with the Agilent G2565BA Microarray Scanner (Agilent Technologies Inc.). The fluorescence intensities of the scanned images were extracted and pre-processed using the Agilent Feature Extraction Software (10.7.3.1).

TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to isolate the total RNA, and its quality was determined using a NanoDrop 2000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Subsequently, an aliquot of total RNA (100 ng) derived from testicular samples was labeled using a Quick Amp Labeling Kit (Agilent Technologies, Santa Clara, CA, USA). Next, each sample was hybridized with an Agilent Gene Expression Hybridization Kit on a Microarray Hybridization Chamber. After washing, the hybrid signal value was scanned with an Agilent G2565BA microarray scanner (Agilent Technologies). The raw data were extracted and then normalized according to quartiles and processed using the limma R package. The genes were analyzed using highly reliable public transcriptome databases (GENCODE, Noncode, LNCipedia, Ensembl, Lncrnadb, and UCSC). LncRNAs and mRNAs with differential expression (NOA vs. control) were identified using Student's t-test with a significance cutoff value of p <  0.05 and an absolute fold-change value > 2.0. The top 20 distinguishable upregulated and downregulated lncRNAs were further displayed by hierarchical clustering. Differences in mRNA expression patterns between samples were illustrated using the heatmap package.

Genome-wide gene expression was analyzed by a SurePrint G3 Human Gene Expression 8 × 60 K v2 Microarray (Agilent Technologies, Santa Clara, CA). cRNA labeled with Cy3 was synthesized from 200 ng of total RNA using a Low Input Quick Amp Labeling Kit (Agilent Technologies), and 600 ng of Cy3-labeled cRNA was fragmented and hybridized to a SurePrint G3 Gene Expression Microarray at 65 °C for 17 hours. Then, the microarray was scanned using an Agilent G2565BA microarray scanner (Agilent Technologies). The obtained signals were processed by Feature Extraction Ver.9.1 (Agilent Technologies), and analyzed by GeneSpring Ver.12.5 (Agilent Technologies). The signal intensity of each probe was normalized so that the 75th percentile of signal intensity of all the probes would be 1.0, and the mean signal intensity of all the probes within a specific gene was used as an expression level of the gene. Gene expression data of mesenchymal stem cells (n = 7, GSE28974) and osteoblasts (n = 3, GSE33382) were obtained from GEO.