Agilent surescan microarray scanner

Total RNA was isolated from A549 cells treated with 50 μM apigenin for 48 h using the miRNeasy mini kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. For miRNA expression analysis, the Human miRNA Microarray V21.0 array (based on miRbase release 21.0, Agilent Technologies, Inc., Santa Clara, CA, USA) was used according to the manufacturer’s protocol. Total RNA was labelled and hybridized using the miRNA Complete Labeling and Hyb Kit (Agilent Technologies, Inc.). The miRNA microarray chips were scanned using an Agilent SureScan MicroArray Scanner (G2600D, Agilent Technologies, Inc.), and the signal values were analyzed using Feature Extraction software 12.0.3.1 (Agilent Technologies, Inc.).

Whole-genome gene expression profiles were analyzed using the Mouse Gene Expression 4x44K v2 Microarray (Agilent Technologies, Tokyo, Japan), which detects 39,430 Entrez Gene RNAs. Total RNA was first isolated with TRIzol reagent (ThermoFisher Scientific, Waltham, MA, USA) from whole brains of four male mice in each group with the Ctrl or Trp− diet. Total RNA was determined using an Agilent 2100 Bioanalyzer. Total RNA was then applied to Cy3-labeled cRNA synthesis, which was performed with a Low Input Quick Amp Labeling Kit (Agilent Technologies) according to the manufacturer’s instructions. Finally, Cy3-labeled cRNA was hybridized to the microarray and detected using an Agilent SureScan Microarray Scanner (Agilent Technologies).
Microarray image data were extracted to ProcessedSignal using Feature Extraction 11.5.1.1 software (Agilent Technologies). GeneSpring GX v14.5 software (Agilent Technologies) was used for subsequent data processing. Gene expression with a statistically significant difference (P < 0.05) and an absolute value of log₂ fold change > 1.2 between groups was exported to the dataset. Gene ontology and pathway analyses were performed using BaseSpace (Illumina KK, Tokyo, Japan) and MetaCore v6.30 build 68780 (Clarivate Analytics Japan, Tokyo, Japan), respectively.

RNA labeling and hybridization were conducted in accordance with the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technologies, V 6.5, 2010, Lexington, MA, USA). Briefly, 200 ng of total RNA from each sample was linearly amplified, and it was labeled with Cy3-dCTP. The labeled cRNAs were purified using an RNeasy Mini Kit (Qiagen, Hilden, Germany). The concentrations and specific activities of labeled cRNAs (pmol Cy3/μg cRNA) were measured using NanoDrop ND-1000 (NanoDrop, Wilmington, NC, USA). Subsequently, 600 ng of each labeled cRNA was fragmented by adding 1 μL of 25 × fragmentation buffer and 5 μL 10 × blocking agent, and they were heated at 60 °C for 30 min. Finally, 25 μL 2 × GE hybridization buffer was added to dilute the labeled cRNA. Following this, 50 μL of hybridization solution was dispensed into the gasket slide, and it was placed in the SurePrint G3 Custom Gene Expression Microarrays, 8 × 60 K (Agilent Technologies). The slides were incubated at 65 °C for 17 h in an Agilent hybridization oven and washed at room temperature according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology, V 6.5, 2010). The hybridized array was immediately scanned using an Agilent SureScan Microarray Scanner (Agilent Technologies).

This procedure was performed using the SurePrint G3 Human CGH + SNP 4 × 180 K Microarray Kit (4 × 180 K) (Agilent Technologies, Santa Clara, CA, USA) with 13 kbp overall median probe spacing (11 kbp in RefSeq genes) according to the manufacturer’s instructions. Labeling and hybridization of the patient’s DNA and reference DNA (#5190-3796, Human Reference DNA male, Agilent Technologies, Santa Clara, CA, USA) were implemented by enzymatic labeling and hybridization protocols (v.7.5, Agilent Technologies, Santa Clara, CA, USA). Array images were acquired on an Agilent SureScan Microarray Scanner (Agilent Technologies, Santa Clara, CA, USA). Data analysis was performed using the CytoGenomics software (v.3.0.6.6) (Agilent Technologies, Santa Clara, CA, USA) and the publicly available Database of Genomic Variants (DGV) resources [23 (link)]. Human genome assembly 19 (hg19) was utilized to describe the molecular karyotype revealed by aCGH.

The high MV or low MV worms were divided and harvested as described in Supplementary Figure 1A. Total RNA was extracted by using miRNeasy mini kit (Qiagen, Cat No. 217004) and was quantified using NanoDrop 2000 (Thermo Fisher Scientific, NanoDrop Products, Wilmington, DE, USA). RNA integrity was assessed using Agilent Bioanalyzer 2100 (Agilent Technologies, CA, USA). The RNA integrity value of all samples is greater than 9. Sample labeling, microarray hybridization, and washing were performed according to the standard protocols of the manufacturer. The total RNA was transcribed to double-stranded complementary DNA (cDNA), synthesized into complementary RNA (cRNA), and finally labeled with cyanine-3-CTP, and then hybridized onto the Agilent C. elegans (V2) Gene Expression Microarray, 4x44K microarray containing 43,803 probes. After washing, the arrays were scanned using an Agilent Surescan microarray scanner (Agilent Technologies). Feature Extraction software (version 11.5.1.1; Agilent Technologies) was used to analyze the array images and obtain raw data.
Microarray expression data that reported in this paper have been deposited in the NCBI Gene Expression Omnibus (GEO) under accession number GSE99020. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE99020.

Gene expression profiling was performed using RNA isolated from 18 snap-frozen MPA/DMBA-induced tumors and six normal/untreated mouse mammary gland samples. Whole genome expression data was obtained using Agilent Sureprint G3 Mouse Gene Expression 8x60K microarrays (Agilent Technologies cat.# G4852B) with Low Input Quick Amp Labeling protocol (Agilent Technologies cat.# 5190-2331) and the Cy3 fluorophore. Forty nanogram RNA was used for input. Microarrays were scanned using an Agilent SureScan Microarray Scanner (Agilent Technologies cat.# G4900DA), and data was extracted using Agilent Feature Extraction software. One tumor sample (S422_15_2) failed quality control and was excluded from further gene expression analyses.

Primary osteoclast precursors were plated at 500,000 cells/well in 6 well plates. Total RNA was isolated from differentiating cultures using the miRNeasy Mini kit (Qiagen, Valencia, CA). On-column DNase treatment was performed to reduce contamination with genomic DNA, and an additional treatment with RQ1 DNase (Promega, Madison, WI) was performed prior to gene expression analysis. RNA concentration and purity were assessed by spectrophotometric analysis. The quality of small RNAs in each sample was determined using the 2100 Bioanalyzer assay (Agilent Technologies, Santa Clara, CA).
Four independent cultures were analyzed for each time point. For each sample, 200 ng of total RNA were labeled using miRNA Microarray System with miRNA Complete Labeling and Hyb Kit (Agilent Technologies). According to the manufacturer’s instructions, the samples were hybridized for 20 hours onto a mouse miRNA Microarray, Release 15.0, 8×15 K (based on Sanger miRBase release 15.0), containing 627 mouse mature miRNA probes (Agilent Technologies). Hybridized and washed array slides were scanned at 5 µm resolution using an Agilent SureScan Microarray Scanner (Agilent Technologies). Image processing was completed using Feature Extraction Software (Agilent Technologies).