BMDMs were stimulated with CpG (0.063 μg/ml), IFN-γ (100 U/ml) + αIL-10R (1.25 μg/ml), or with all three agents at 37 °C for 20 h. Total RNA from each BMDM sample was then extracted using RNeasy Plus Mini Kits (QIAGEN) according to the manufacturer's instructions. Microarray analyses were performed by MBL, Japan, using SurePrint G3 Mouse Gene Expression 8 × 60 K Microarrays (Agilent). For qPCR analyses, RNA was reverse transcribed using ReverTra Ace qPCR master mix (TOYOBO, Japan), and cDNA products were amplified using a LightCycler 96 (Roche) with Universal SYBR Select master mix (Thermofisher). Data were analyzed using the delta Ct method and were normalized to β-actin RNA expression in each sample. The original microarray data were deposited in GEO database under accession number GSE90881 . The primers for real-time PCR were as follows: β-actin-Fw, 5′-CTTTGCAGCTCCTTCGTTG-3′ and β-actin-Rv, 5′-CGATGGAGGGGAATACAGC-3′; ICAM-1-Fw, 5′-GTCCGCTGTGCTTTGAGAAC-3′ and ICAM-1-Rv, 5′-TGAGGTCCTTGCCTACTTGC-3′; VCAM-1-Fw, 5′-GAAATGCCACCCTCACCTTA-3′ and VCAM-1-Rv, 5′-CGGGGGAGATGTCAACAATA-3′; LFA-1α-Fw, 5′-CAGAACAAGAACCCCGATGT-3′ and LFA-1α-Rv, 5′-CCTGGCACCAGACTCTTCTT-3′; and VLA-4α-Fw, 5′-AATGCCTCAGTGGTCAATCC-3′ and VLA-4α-Rv, 5′- TCTCCTCCAGGCATGTCTTC-3′.
Sureprint g3 mouse gene expression 8 60 k microarray
Manufactured by Agilent Technologies
Sourced in United States
The SurePrint G3 Mouse Gene Expression 8 × 60 K Microarray is a high-density microarray designed for comprehensive gene expression analysis of the mouse genome. It features 60,000 mouse probes, covering over 39,000 mouse genes, providing a comprehensive view of the mouse transcriptome.
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Lab products found in correlation
Low input quick amp labeling kit, by Agilent Technologies (4 mentions)
Bioanalyzer, by Agilent Technologies (2 mentions)
Feature extraction software, by Agilent Technologies (2 mentions)
Universal sybr select master mix, by Thermo Fisher Scientific (1 mentions)
Lightcycler 96, by Roche (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Revertra ace qpcr master mix, by Toyobo (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Dnase set, by Qiagen (1 mentions)
Feature extraction version 10.10.1.1, by Agilent Technologies (1 mentions)
One color microarray based gene expression analysis low input quick amp labeling, by Agilent Technologies (1 mentions)
Evagreen, by Biotium (1 mentions)
Gdna remover, by Toyobo (1 mentions)
G2565 microarray scanner, by Agilent Technologies (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Rneasy minelute cleanup kit, by Qiagen (1 mentions)
Amplitaq gold 360 master mix, by Thermo Fisher Scientific (1 mentions)
Revertra ace qpcr rt master mix, by Toyobo (1 mentions)
Feature extraction 10, by Agilent Technologies (1 mentions)
10 protocols using sureprint g3 mouse gene expression 8 60 k microarray
1
Transcriptomic Analysis of Macrophage Activation
2
Integrative Microarray Data Analysis
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Normalized microarrays from seven studies (GSE84185, GSE43261, GSE56028, GSE118669, GSE54307, GSE6476, and GSE42940) were retrieved from GEO using GEOquery [160 (link)]. One last microarray was retrieved directly from the authors [50 (link)] and was normalized using RMA from the oligo R package [161 (link)]. The microarrays’ latest annotations were retrieved from Ensembl Biomart for Affymetrix GeneChip Mouse Genome 430 2.0, Affymetrix GeneChip Mouse 430A 2.0, Affymetrix Rat Gene 1.0 ST arrays, and Agilent SurePrint G3 Mouse Gene Expression 8 × 60 K microarrays. When unavailable, annotations from the original authors were completed using megablast queries (GSE42940). Probes with mapping to multiple genes were discarded. Differential gene expression analyses were produced with Limma [162 (link)].
3
Mouse Kidney RNA Isolation and Microarray Analysis
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Total RNA from mouse kidneys was extracted using TRIzol Reagent (Invitrogen) and purified with RNase-free DNase Sets and RNeasy Kits (Qiagen) according to the manufacturer’s protocol. The microarray experiments were performed using SurePrint G3 Mouse Gene Expression 8 × 60 K microarrays (Agilent Technologies). Microarray data files can be obtained from the NIH Gene Expression Omnibus with accession number GSE87600. The data were analyzed by the National Institute on Aging (NIA) microarray analysis tool (http://lgsun.grc.nia.nih.gov/ANOVA/ )49 (link). Bioinformatics analysis for the gene profile of cytokines and cytokine receptors was performed using the DAVID Functional Annotation Bioinformatics Microarray Analysis (https://david-d.ncifcrf.gov/ )50 (link)51 (link).
4
Microarray Analysis of Rfx6 Knockout Mice
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Samples processing: Microarray analysis was performed according to Agilent protocol “One-Color Microarray-Based Gene Expression Analysis – Low Input Quick Amp Labeling” version 6.5, May 2010 (Cat #G4140-90040). Complementary RNA (cRNA) samples were linearly amplified and labeled with cyanine 3 starting from 100 ng of total RNA. Following fragmentation, labeled cRNA were hybridized on Agilent “SurePrint G3 Mouse Gene Expression 8 × 60 K Microarray” (Design ID: 028005), for 17 h, at 65 °C under 10 rpm. Following washing, the slides were scanned using an Agilent G2565CA microarray Scanner System, at a 3 μm resolution in a 20-bit scan mode, according to the “AgilentG3_GX_1Color” protocol. Data analysis: Raw.tif images were then extracted using Agilent “Feature Extraction, version 10.10.1.1” following “GE1_1010_Sep10” protocol. Median raw expression values were further normalized by quantile method [39] (link). Differential expression analysis between Rfx6−/− vs controls and Rfx6ΔEndo vs controls were performed using the R package limma version 3.36.5 [40] (link). Data have been deposited in GEO repository (GSE133038).
5
Transcriptome Analysis of Tissue Samples
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Total RNA was extracted from tissue or cell samples using AGPC methods and was used as a template for cDNA synthesis with the ReverTra Ace qPCR RT Master Mix and gDNA Remover (Toyobo). qPCR was performed using Prism 7900HT or Step One Plus Real-Time PCR Systems (Thermo Fisher) with AmpliTaq Gold® 360 Master Mix (Thermo Fisher), EvaGreen (Biotium), and the primers listed in Table S1 ; 18S rRNA served as an internal control. Prior to the microarray analysis, a Low Input Quick Amp Labeling Kit (Agilent Technologies) was used to label total RNA that had been purified using the RNeasy MinElute Cleanup Kit (Qiagen). The labeled RNA was then used to probe a SurePrint G3 Mouse Gene Expression 8 × 60K Microarray (Agilent Technologies), and the signals were scanned using a G2565 microarray scanner (Agilent Technologies). Microarray data were extracted from the scanned images using Feature Extraction 10.7 software (Agilent Technologies), and the raw unfiltered microarray data were deposited in the Gene Expression Omnibus dataset (subseries entries GSE149423 ). The differentially expressed genes were isolated and analyzed using the Subio Platform (Subio).
6
RNA Isolation, Quality Assessment, and Gene Expression Analysis
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Total RNA was isolated from the indicated tumor cell lines with RNeasy® Mini Kit (Qiagen, Germany) according to the manufacturer’s protocol. The sample quality was analyzed using a bioanalyzer (Agilent Technologies, Santa Clara, USA) with the RNA6000 Nano kit (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s guidelines. Samples with an RIN (RNA integrity number) higher than 7.0 were used for further analysis. Gene expression analysis was carried out using the SurePrint G3 Mouse Gene Expression 8 × 60 K Microarray (Design ID 028005; Agilent Technologies, Santa Clara, CA, USA). Samples were labeled with the Low Input Quick Amp Labeling Kit (Agilent Technologies) according to the manufacturer’s guidelines. Slides were scanned in a G2565CA microarray scanner and extracted with the Feature Extraction Software (v.10.7.3.1, Agilent Technologies).
7
Duodenal RNA Expression Analysis
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Total RNA was isolated from fresh frozen duodenal tissue from control (vehicle gavage, n = 6; control chow, n = 3) and G007-LK treated (10 mg/kg gavage, n = 5; 50 mg/kg gavage, n = 2; 100 mg/kg chow, n = 3) mice. 40 ng total RNA was used in the Low Input Quick Amp Labeling protocol (Agilent Technologies) before hybridisation to the SurePrint G3 Mouse Gene Expression 8 × 60K Microarray (Agilent Technologies) according to the manufacturer’s protocol. Data were pre-processed and analysed using GeneSpring GX software, version 13.0 (Agilent Technologies). In brief, the mRNA probes were normalised to the 75 percentile and median-centred. The normalised data were filtered based on expression (20–100 percentile). Unsupervised hierarchical clustering was performed using the Euclidean algorithm for similarity measures and Ward’s rule for linkage.
8
Microarray Analysis of Gene Expression
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Total RNA was isolated from five individual cell lines. The quality was analyzed with a bioanalyzer (Agilent Technologies, Santa Clara, USA). Gene expression analysis was carried out using the SurePrint G3 Mouse Gene Expression 8×60K Microarray (Design ID 028005; Agilent Technologies). Samples were labeled with the Low Input Quick Amp Labeling Kit (Agilent Technologies) according to the manufacturer’s guidelines. Slides were scanned using a G2565CA microarray scanner (Agilent Technologies). Raw data files were extracted using the Feature Extraction software (v.10.7.3.1, Agilent Technologies). Raw microarray data were background corrected and quantile normalized using the R and Bioconductor package limma (https://www.R-project.org ).31 32 After filtering control probes and low expressed probes, differential expression analysis was performed using limma.
9
Microarray Analysis of DGKη-KO Mice
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Cerebral cortexes were homogenized in QIAzol lysis reagent (Qiagen, Hilden, Germany), and total RNA was isolated with the Direct-zol™ RNA Miniprep (ZYMO RESEARCH, Irvine, CA). Microarray hybridization (SurePrint G3 Mouse Gene Expression 8 × 60K Microarray, Agilent, Santa Clara, CA, USA) was performed at the TaKaRa Bio (Kusatsu, Japan). Three experiments using independent cohorts were carried out.
Differential gene expression in DGKη-KO mice was normalized to WT data (WT = 1 (log21 = 0)) to obtain the fold-change values of all genes. The values are presented as the mean (log2) ± SEM of three independent experiments. Statistical comparisons were performed using Student's t-tests. A cut-off P-value <0.05 and fold-change ≥ absolute 2 (log22 = 1 or log21/2 = −1) were applied.
Differential gene expression in DGKη-KO mice was normalized to WT data (WT = 1 (log21 = 0)) to obtain the fold-change values of all genes. The values are presented as the mean (log2) ± SEM of three independent experiments. Statistical comparisons were performed using Student's t-tests. A cut-off P-value <0.05 and fold-change ≥ absolute 2 (log22 = 1 or log21/2 = −1) were applied.
10
Microarray Analysis of Arp5, Ies6, and Ino80 Knockdown in RD Cells
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Total RNAs were isolated from RD cells transfected with control, Arp5, Ies6, or Ino80 siRNA for 2 days, using NucleoSpin RNA Plus (TAKARA BIO). RNAs from three independent experiments (biological replicates) were mixed in equal amounts and used as samples for subsequent microarray analysis. mRNAs were reverse-transcribed, and Cy3-labeled complementary RNAs (cRNAs) were synthesized using the Low Input Quick Amp Labeling Kit (Agilent Technologies). The cRNAs were hybridized on a SurePrint G3 Mouse Gene Expression 8 × 60 K Microarray (Agilent Technologies), and fluorescence signals were detected using the SureScan Microarray Scanner (Agilent Technologies). The fluorescence intensity was quantified using Feature Extraction Software (Agilent Technologies).
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