RNA derived from three different pooled samples at 3 μg/25 μl was analyzed by Genome Québec. Mouse Gene ST 2.0 arrays from Affymetrix were used for mRNA and GeneChip miRNA 4.0 (Affymetrix) Array (Mouse) for miRNAs. Microarray readouts were downloaded from Genome Québec server and analyzed with Expression Console (EC) software (Affymetrix) to assess quality metrics such as absolute deviation residuals, relative log expression and pos_neg_AUC to estimate the correct positive rate. After a quality assessment, samples were analyzed with the Transcriptome Analysis Console (TAC 3.0, Affymetrix). S1 Data shows all genes and miRNAs analyzed in Mouse Gene ST 2.0 and GeneChip miRNA 4.0 at 6 and 24 hpi.
Mouse gene st 2.0 arrays
Manufactured by Thermo Fisher Scientific
The Mouse Gene ST 2.0 arrays from Thermo Fisher Scientific are high-density oligonucleotide microarrays designed for gene expression analysis of the mouse genome. The arrays provide comprehensive coverage of the mouse transcriptome, enabling the measurement of the expression levels of protein-coding and non-coding genes.
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Lab products found in correlation
Genechip scanner 3000 7g, by DNASTAR (2 mentions)
Expression and transcriptome analysis console software, by Thermo Fisher Scientific (2 mentions)
Genechip mirna 4, by Thermo Fisher Scientific (1 mentions)
Expression console software, by Thermo Fisher Scientific (1 mentions)
Rt2sybr green qrt pcr kit, by Bio-Rad (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Wt expression kit, by Thermo Fisher Scientific (1 mentions)
Ingenuity pathway analysis software, by Qiagen (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Applause wt amp plus st system, by Tecan (1 mentions)
Peqgold trifast, by Avantor (1 mentions)
Genechip scanner 3000 7g, by Thermo Fisher Scientific (1 mentions)
10 protocols using mouse gene st 2.0 arrays
1
Analyzing Gene Expression in Mouse Samples
2
Transcriptome Analysis of Osteochondrogenic Progenitors
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Total RNA was extracted using RNeasy kit (Qiagen) from short-term-expanded and FACS-enriched primary OCPs and their derivatives from 3-day-old SHP2Prrx1CTR;R26mTmG and SHP2Prrx1KO;R26mTmG mice, and analyzed for integrity by using an Agilent 2100 Bioanalyzer. For differential gene expression analysis, three RNA samples per mouse line were amplified using Invitrogen WT Expression kit and hybridized to Affymetrix Mouse Gene ST 2.0 arrays. Ingenuity Pathway Analysis (IPA) Software from Qiagen was used for pathway analysis.
To validate the expression of differentially regulated genes on arrays, qRT-PCR was performed with RT2SYBR®Green qRT-PCR kit on a Bio-Rad CFX machine using cDNA that was synthesized using 1 µg total RNA with iScript™cDNA Synthesis Kit (Bio-Rad). All samples were normalized to Gapdh and gene expression data were presented as fold-increases or -decreases compared with controls. All primer sequences used for this study are available by request.
To validate the expression of differentially regulated genes on arrays, qRT-PCR was performed with RT2SYBR®Green qRT-PCR kit on a Bio-Rad CFX machine using cDNA that was synthesized using 1 µg total RNA with iScript™cDNA Synthesis Kit (Bio-Rad). All samples were normalized to Gapdh and gene expression data were presented as fold-increases or -decreases compared with controls. All primer sequences used for this study are available by request.
3
Differential Keratinocyte Gene Expression
4
Femur Gene Expression Analysis in BXD Mice
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The expression data was generated from both parents, C57BL/6J (B6) and DBA/2J (D2) and 34 BXD strains. Briefly, femurs were collected free of soft tissue observed by naked eye from each mouse. Bones were then cleared off of the surrounding connective tissue. RNA was isolated from the femur of BXDs and hybridized onto Affymetrix Mouse Gene ST 2.0 arrays. The raw data was first normalized using the robust-multichip array (RMA) method [27 (link)], and then z-score normalized. But, instead of leaving the mean at 0 and the standard deviation of 1 unit, the data was rescaled to a mean of 8 units with a standard deviation of 2 units (also known as 2Z + 8 normalized data). The normalized data can be accessed through our GeneNetwork portal (http://genenetwork.org/ ) [28 ] with the name “UTHSC WGU88 Female Bone Femur AFFY Mouse Gene ST 2.0 Gene Level (Oct13) RMA” (GeneNetwork ID: GN466, https://files.genenetwork.org/current/GN466/ ), under the group name “BXD Family” and type “Bone Femur mRNA”. All experimental procedures were in accordance with the Guidelines for the Care and Use of Laboratory Animals published by the National Institutes of Health and were approved by the Animal Care and Use Committee at the University of Tennessee Health Science Center (UTHSC; Memphis, TN, USA. Approval number: 21–0306).
5
Transcriptome Profiling of Splenocytes
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RNA was isolated from splenocytes, using guanidinium thiocyanate-phenol-chloroform extraction (Peqgold Trifast, Peqlab). cDNA was synthesized and quality controlled using the Applause WT-Amp Plus ST System (NuGEN), according to the manufacturer’s protocol. Mouse Gene ST 2.0 arrays (Affymetrix) were performed by standard protocol and read on a Gene Chip Scanner 3000 7G (Affymetrix).
To identify differentially expressed genes (DEGs), we employed thresholds of p ≤ 0.05 (Student’s paired t-test) and an absolute log2 fold change in expression ≥0.3 for each gene (probe set). To interpret the biological function of DEGs, we performed KEGG pathway enrichment analysis based on a hypergeometric test, which was done by the R package GOstats26 (link).
To identify differentially expressed genes (DEGs), we employed thresholds of p ≤ 0.05 (Student’s paired t-test) and an absolute log2 fold change in expression ≥0.3 for each gene (probe set). To interpret the biological function of DEGs, we performed KEGG pathway enrichment analysis based on a hypergeometric test, which was done by the R package GOstats26 (link).
6
Mouse Transcriptomic Profiling by Microarray
7
Differential Keratinocyte Gene Expression
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RNA isolated from 4 independent pooled Ahr+/+ primary keratinocyte cultures and 3 Ahr-/- cultures were reverse transcribed and hybridized to Affymetrix Mouse Gene ST 2.0 arrays in the Penn State Genomics Core Facility according to the manufacturers protocol. Arrays were scanned using a GeneChip Scanner 3000 7G and analyzed using ArrayStar 11 Software (DNASTAR, Madison WI) with RMA background correction and quantile normalization. Mean log2 signal was used to compare gene expression between groups and significantly different genes identified using a 1.5-fold cut off and p value <.05 using a one sided equal variance Student t test. Functional annotation clustering with genes identified as differentially expressed using ArrayStar11 was conducted using DAVID Bioinformatics Software (Huang et al., 2009a (link); Huang et al., 2009b (link)). GEO accession number:GSE62490.
8
Mouse Transcriptomic Profiling by Microarray
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RNA samples were isolated from age-matched mice of different genotypes. Fluorescently-labeled cDNA probes were prepared from extracted RNA samples and hybridized to Affymetrix Mouse Gene ST 2.0 arrays. Array hybridization, washing, and scanning were carried out per the manufacturer's instructions. Data processing and analysis were performed with Affymetrix Expression and Transcriptome Analysis Console software.
9
Profiling Ninj1-Deficient Macrophage Transcriptome
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RNA was isolated from peritoneal macrophages extracted from WT and Ninj1 KO mice. RNA from each sample was synthesized to make cDNA by using the GeneChip Whole Transcript amplification kit, according to the manufacturer’s protocol. The cDNA was hybridized with the Affymetrix Mouse Gene ST 2.0 arrays and analyzed by Macrogen (Seoul, Korea). Data were normalized by applying the multi-average (RMA) method, and statistical significance was calculated by applying the LPE test.
10
Colonic Transcriptomic Profiling of Ptch1 Mice
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RNA was isolated from 1 cm of fresh-frozen whole colonic tissue from Col1a2CreER;Ptch1fl/fl mice (n=4) and controls (n=4) starting 1.5 cm proximal to the anus. RNA quality was assessed using the Agilent 2200 Tape Station system; RNA integrity numbers >8 were considered sufficient. Affymetrix Mouse Gene ST 2.0 arrays were used. Robust multi-array average median polish procedures were applied for normalization. For GSEA analyses58 (link), the GSEA Java plug-in v2.1.0 was used to probe non-log-transformed normalized expression data with standard settings (with the exception that the permutation type was set to ‘gene_set' and gene sets <15 genes were permitted). Gene lists were derived either from publications as indicated, from the Molecular Signature Database (www.broadinstitute.org/gsea/msigdb ), or from gene ontology searches via the AmiGO2 database v 2.1.4 (www.geneontology.org ), curated to include ‘manual assertion' and ‘experimental evidence', and excluding ‘sequence similarity evidence' (see also Supplementary Tables 2–5 for the gene sets used).
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