One color microarray based gene expression analysis

Analysis of an Agilent 4 × 180 k custom oligoarray of triplicate samples was performed as described (Wang et al., 2018). Total RNA was extracted with an ISOLATE II RNA kit (Bioline, Eveleigh, Australia). RNA samples were screened with an Agilent Bioanalyzer to ensure the RNA was of high quality, and 100 ng was amplified and labeled following the manufacturer's instructions for One‐Color Microarray‐based Gene Expression Analysis (Agilent, Mulgrave, Australia). Microarray data were processed with Agilent's feature extraction software (v.10.7), and differential expression was determined using a Bayesian adjusted t‐statistic within the ‘Linear Models for Microarray Data’ r‐package. P values were corrected for a false discovery rate (FDR) of 5%. Probes with a fold change (FC) of ≥ 1.5/≤ −1.5 and FDR corrected P ≤ 0.05 between two groups were defined as significantly different.

The RNA derived from five different MSC samples exposed to one cycle of QMR stimulation and their corresponding MSC controls were extracted using RNeasy plus mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The total RNA quantification was obtained with NanoDrop UV-VIS Spectrophotometer (Thermo Fisher Scientific). The quality of RNA was determined using Agilent 2100 Bioanalyzer system with Eukaryote Total RNA Nano kit (Agilent Technologies, Santa Clara, CA, USA). The samples were processed according to protocol “Agilent One-Color Microarray-based Gene Expression Analysis (Low Input Quick Amp Labeling)” with Human GE 4x44K V2 Microarray Kit (Agilent Technologies).
Microarray slides were detected with Agilent scanner through ScanControl software. Row data from microarray images were extracted by Agilent Feature Extraction software. Afterward data were subjected to a pre-processing step using open-source program Bioconductor that employs the Limma package with R language [30 (link)].

Total RNA was isolated from 9 Rb tumors and 2 control pediatric retina samples using an Agilent Absolutely RNA miRNA kit (cat# 400814, Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s instructions. The quality of the isolated RNA was determined on an Agilent 2200 TapeStation system (cat#G2964AA, Agilent Technologies, Santa Clara, CA, USA) using an Agilent RNA ScreenTape assay (cat#5067-5576, Agilent Technologies, Santa Clara, CA, USA). mRNA labeling and microarray processing was performed as detailed below in the “One-Color Microarray-Based Gene Expression Analysis” (cat# G4140-90040, Agilent Technologies, Santa Clara, CA, USA). miRNA labeling was done using an Agilent miRNA Complete Labeling and Hybridization Kit (Cat# 5190-0456, Agilent Technologies, Santa Clara, CA, USA). The gene expression and miRNA data were extracted using Agilent Feature Extraction Software (11.5.1.1) and analyzed using Agilent GeneSpring GX 13.1. The analysis was carried out using a t-test unpaired statistical method with the Benjamini–Hochberg FDR method. In both the mRNA and miRNA analyses, transcripts exhibiting p ≤ 0.05 and fold changes greater than or equal to two were considered differentially expressed entities. Both the mRNA (GSE208143) and miRNA (GSE208677) microarray data were submitted to the NCBI GEO database.

For the microarray analysis, total RNA from rat hippocampal neuronal cultures subjected to control or OGD conditions was collected after 7 h and 24 h of post-incubation in culture conditioned medium. RNA from three independent cultures was used as biological replicates. Equal amounts of RNA extract (200 ng) from each replicate were amplified and Cy-3-labeled using the Low Input Quick Amp Labeling kit (Agilent Technologies, Santa Clara, CA). Hybridizations were carried out following Agilent Technologies instructions for One-Color Microarray-Based Gene Expression Analysis (Agilent Technologies), using whole-genome Rat GE 4×44K v3 Microarrays. Images were obtained using the Agilent G2565AA microarray scanner and fluorescence quantization was performed using the Agilent Feature Extraction 10.5.1.1 software and the GE1_105_Dec08 protocol. The signal intensity was aligned and normalized between microarrays by centering the median of the signal distribution using BRB-ArrayTools v3.8.1. The microarray data was submitted to GEO database and has been given the accession number GSE54037.

The quantity and the quality of extracted RNAs were assessed using a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, Eclubens, Switzerland) and 2100 Bioanalyzer (Agilent Technologies, Montpellier, France). Microarray experiments used chips containing 45.000 probes (4x44K Whole Human Genome) and One-Color Microarray-Based Gene Expression Analysis provided by Agilent Technologies. Four hundred ng of extracted RNAs were labeled using Cyanin-3 CTP using a low input Quick Amp Labeling kit, one color (Agilent technologies). This step lead to amplify and label target RNA to generate cRNA (Cy3-) for further oligo microarrays used in gene expression profiling. Labelled RNAs were hybridized for 17 hours at 65°C using QIAmp labeling kit according to the manufacturer’s recommendations. Slides were washed and scanned with a pixel size of 5 μm using a DNA Microarray scanner G2505C. The raw data were extracted using feature Extraction Software 10.5.1. Data were processed using GeneSpring GX 14.9 software (Agilent Technologies, Montpellier, France). Modulated genes were analyzed using Ingenuity Pathway Analysis (IPA, Qiagen, Courtaboeuf, France) or ClustVis software. The data have been deposited in NCBI’s Gene Expression Omnibus and are accessible via GEO series accession number GSE (GSE112086).

RNA integrity was confirmed with a Bioanalyzer 2100 (Agilent Technologies). A custom Agilent gene expression microarray was used (Comadira et al., 2015 (link)). Microarrays were processed according to the ‘One-Color Microarray-Based Gene Expression Analysis’ protocol (v. 6.5; Agilent Technologies). Experimental design and complete datasets have been deposited in the ArrayExpress database at EMBL-EBI (www.ebi.ac.uk/arrayexpress) under accession numbers E-MTAB-7228, E-MTAB-7229, E-MTAB-7230, E-MTAB-7231. Data were extracted using Feature Extraction (FE) software (v. 10.7.3.1; Agilent Technologies) with default settings, and subsequently processed using GeneSpring GX (v. 7.3; Agilent Technologies) software. Data were normalised using Agilent FE one-colour settings: for each experiment, data were set to a minimum of 5 and normalised within each array to the 50th percentile of raw expression values, and individual probe data was subsequently normalised to its median value across all arrays. Flag-filtered data quality was visually assessed using box plots and performing Principal Component Analysis (PCA) for all replicates in each tissue sample using the R package ‘FactoMineR’ (Husson et al., 2016 ).