RNA extraction was performed using the RNAqueous-Micro Kit (Ambion), amplified using the Ovation Pico WTA Sytem (NuGEN), and hybridized to GeneChip Mouse Gene 2.0 ST microarrays (Affymetrix). Fragmentation was performed with the NuGen Encore biotin module and samples were hybridized in a GeneChip Hybridization Oven 640 for 18 h at 45 °C. Data normalization was performed using robust multiarray average (RMA) summarization and quartile normalization using ArrayStar software (DNASTAR). CDP and CD24+ cDC expression values were averaged from biological triplicates, and all other expression values were averaged from biological duplicates. Principal component analysis was computed by singular value decomposition without additional centering or scaling after samples were grouped by replicate and the mean log-transformed expression values from each group imported into R, mean-centered by gene, root mean square (RMS)-scaled by sample, and transposed. Unsupervised hierarchical clustering of differentially expressed genes was computed using ArrayStar (DNAstar) with a Euclidean distance metric and centroid linkage method.
Genechip mouse gene 2.0 st microarrays
Manufactured by Thermo Fisher Scientific
Sourced in United States
The GeneChip Mouse Gene 2.0 ST microarrays are a tool for gene expression analysis in mouse models. The microarrays provide comprehensive coverage of the mouse transcriptome, allowing for the detection and quantification of mRNA levels for over 35,000 well-annotated mouse genes.
Automatically generated - may contain errors
Lab products found in correlation
Encore biotin module, by Tecan (2 mentions)
Rnaqueous micro kit, by Thermo Fisher Scientific (2 mentions)
Ovation pico wta sytem, by Tecan (2 mentions)
Genechip hybridization oven 640, by Tecan (2 mentions)
Bioanalyzer, by Agilent Technologies (2 mentions)
Taqman gene expression array, by Thermo Fisher Scientific (1 mentions)
Rneasy minelute kit, by Qiagen (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Genechip scanner 3000 7g, by Thermo Fisher Scientific (1 mentions)
Rneasy plus micro kit, by Qiagen (1 mentions)
5 protocols using genechip mouse gene 2.0 st microarrays
1
Microarray Analysis of Dendritic Cell Subsets
2
Microarray Analysis of Dendritic Cell Subsets
3
Transcriptional Profiling of Donor PECs
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Total RNA was isolated (Qiagen) from total peritoneal exudate cells (PECs), magnetic bead-isolated CD115+ PECs, or sorted CD115+ CD45.1+ donor PECs, from day 4 AIP. Individual gene expression was measured by RT-PCR using TaqMan Gene Expression Arrays (ThermoFisher). For transcriptomic analysis, RNA was quantified, normalised and verified by Bioanalyzer (Agilent) prior to processing onto Genechip Mouse Gene 2.0ST Microarrays (Affymetrix). Sorted CD115+ donor PECs additionally underwent PCR amplification prior to analysis (Nugen). Normalisation across all arrays was achieved using the robust multi-array average (RMA) expression measure [34 (link)] which results in expression measures (summarised intensities) in log base 2. Significant genes from each comparison were analysed for enrichment of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway membership using a hypergeometric test. Pathway enrichment (p < 0.05) was assessed separately for upregulated and downregulated genes.
4
Microarray Analysis of Dendritic Cells
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Total RNA was extracted from up to 50,000 DCs per subset using Trizol (Invitrogen) phase separation and purified using the RNeasy MinElute kit (Qiagen, Hilden, Germany). RNA integrity was measured using a BioAnalyzer (Agilent, Santa Clara, CA, USA) and the mean RNA integrity score +/− SD was 9.5 +/− 0.5 for all samples. RNA samples were shipped on dry ice to the Ramiciotti Center for Genomics (New South Wales, Australia) where sample amplification was performed with the Ovation Pico WTA v2 protocol (NuGen, San Carlos, CA, USA). Hybridisation to GeneChip™ Mouse Gene 2.0 ST microarrays (Affymetrix, Santa Clara, CA, USA) was performed according to standard procedures.
5
Transcriptome Profiling of Mouse Tissue
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