Total RNA was isolated with Trizol reagent (Invitrogen, 15596-018) following the manufacturer's instructions either directly (Oct4 experiments) or after trypsinization (Tbx3 experiments). 250ng of total RNA was amplified to generate labeled cRNA using the Illumina Total Prep RNA Amplification Kit (Ambion, AMIL1791). 750ng of each labeled cRNA sample was hybridized to MouseRef-8 v2 Expression BeadChip (Illumina, BD-202-0202) and the chip scanned on an Illumina BeadArray Reader. Raw probe level data were exported from Illumina Bead Studio for further processing in R (www.r-project.org ). Raw probe level intensity values were adjusted using the R package limma29 to apply quantile normalization, background subtraction and to transform to log2 expression values. To identify differentially expressed genes, probes were ranked by highest difference in log2 expression value between any two populations. Genes with log fold changes greater than 3 were used for the generation of hierarchically clustered heatmaps. Data are displayed relative to the average log2 expression signal in dCas9-effector Ctrl-sgRNA samples. In instances where multiple probes mapped to the same gene, only expression values for the highest ranked probe were retained. All gene expression data are available through GEO under accession number GSE64059.
Mouseref 8 v2 expression beadchip
Manufactured by Illumina
Sourced in United States
The MouseRef-8 v2 Expression BeadChip is a lab equipment product designed for gene expression analysis. It provides a platform for measuring the expression levels of known mouse genes across multiple samples simultaneously.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (5 mentions)
Beadarray reader, by Illumina (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Amersham fluorolink streptavidin cy3, by GE Healthcare (2 mentions)
Bead array reader confocal scanner, by Illumina (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Genomestudio v2009, by Illumina (1 mentions)
Nextbio, by Illumina (1 mentions)
Agilent bioanalyzer, by Agilent Technologies (1 mentions)
Genomestudio v2009.2 gene expression module v1 5 4, by Illumina (1 mentions)
Lps o111 b4, by Merck Group (1 mentions)
Genomestudio v2011, by Illumina (1 mentions)
Dnase 1, by Qiagen (1 mentions)
Matlab, by MathWorks (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Rnalater, by Qiagen (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Bioanalyzer nanochip, by Agilent Technologies (1 mentions)
Iscan array scanner, by Illumina (1 mentions)
13 protocols using mouseref 8 v2 expression beadchip
1
Transcriptional Profiling of Stem Cell Genes
2
Transcriptional Profiling of Stem Cell Genes
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Total RNA was isolated with Trizol reagent (Invitrogen, 15596-018) following the manufacturer's instructions either directly (Oct4 experiments) or after trypsinization (Tbx3 experiments). 250ng of total RNA was amplified to generate labeled cRNA using the Illumina Total Prep RNA Amplification Kit (Ambion, AMIL1791). 750ng of each labeled cRNA sample was hybridized to MouseRef-8 v2 Expression BeadChip (Illumina, BD-202-0202) and the chip scanned on an Illumina BeadArray Reader. Raw probe level data were exported from Illumina Bead Studio for further processing in R (www.r-project.org ). Raw probe level intensity values were adjusted using the R package limma29 to apply quantile normalization, background subtraction and to transform to log2 expression values. To identify differentially expressed genes, probes were ranked by highest difference in log2 expression value between any two populations. Genes with log fold changes greater than 3 were used for the generation of hierarchically clustered heatmaps. Data are displayed relative to the average log2 expression signal in dCas9-effector Ctrl-sgRNA samples. In instances where multiple probes mapped to the same gene, only expression values for the highest ranked probe were retained. All gene expression data are available through GEO under accession number GSE64059.
3
Illumina MouseRef-8 Expression Profiling
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The labeled cRNA samples (0.75 μg) were hybridized to the Illumina MouseRef-8 v2 expression BeadChip (Illumina, Inc., San Diego, USA) for 16–18 h at 58 °C, according to the manufacturer’s instructions. Detection of the array signals was carried out using Amersham Fluorolink Streptavidin-Cy3 (GE Healthcare Bio-Sciences, Little Chalfont, UK), following the bead array manual. Arrays were scanned with an Illumina bead array reader confocal scanner. Array data analysis was performed using Illumina Genome Studio v.2009.2 (Gene Expression Module v.1.5.4).
4
Mef2c-associated Gene Expression Analysis
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Total RNA was extracted from frozen tissues prepared from the hippocampi of WT and Mef2c-het mice at postnatal day 30, using the Qiagen miRNA kit. RNA concentrations were determined using a Nanodrop spectrophotometer (Thermo Fisher Scientific), and RNA quality was assessed using an Agilent Bioanalyzer. All RNA samples included in the expression analysis had an RNA integrity number (RIN) >8. MouseRef-8 v2 expression beadchip (Illumina) was used for the gene-expression microarray. Microarray data analysis was performed using the R software and Bioconductor packages. Raw expression data were log2 transformed and normalized by quantile normalization. Data quality control criteria included high inter-array correlation (Pearson correlation coefficients >0.85) and detection of outlier arrays based on mean inter-array correlation and hierarchical clustering.
For pathway enrichment analysis, all genes whose expression was statistically altered (P < 0.05) in Mef2c-het mice relative to WT mice were clustered for GO terms using the pathway enrichment application of NextBio (Illumina, Inc.). The background set of genes used was the entire human genome. Rank scores were assigned by NextBio53 (link). Genes clustered to GO terms related to neuronal development were prioritized for validation of changes in gene expression.
For pathway enrichment analysis, all genes whose expression was statistically altered (P < 0.05) in Mef2c-het mice relative to WT mice were clustered for GO terms using the pathway enrichment application of NextBio (Illumina, Inc.). The background set of genes used was the entire human genome. Rank scores were assigned by NextBio53 (link). Genes clustered to GO terms related to neuronal development were prioritized for validation of changes in gene expression.
5
Microarray Analysis of LPS-Stimulated Splenocytes
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For microarray analysis, we stimulated the splenocytes from WT and tIK-Tg mice with 500 ng/ml LPS O111:B4 (Sigma). The Illumina MouseRef-8 v2 Expression BeadChip was used for transcript profiling; 750 ng of labeled cDNA samples were hybridized to each chip and scanned with an Illumina Bead Array Reader confocal scanner, according to the manufacturer’s instructions (Illumina, Inc., USA). The quality of hybridization and overall chip performance were monitored by visual inspection of both internal quality control checks and the raw scanned data. Raw data were extracted using the software provided by the Illumina GenomeStudio v2009.2 (Gene Expression Module v1.5.4). Array data were filtered by detection p-value < 0.05 (similar to signal-to-noise) in at least 50% of samples. (We applied a filtering criterion for data analysis; a higher signal value was required to obtain a detection p-value < 0.05). The selected gene signal value was logarithm-transformed and normalized by the quantile method. The comparative analysis between test and control samples was performed using fold changes. Microarray data for mRNA were verified by real-time PCR.
6
Transcriptome Analysis of Mouse Samples
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Total RNA was prepared with the RNeasy Mini Kit (QIAGEN) according to the manufacturer's instructions and was subsequently processed to yield biotinylated cRNA using the Ambion Illumina RNA amplification kit (Ambion, Austin, TX, USA) according to the manufacturer's instructions. The labeled cRNA preparations were applied to a MouseRef-8 v2 Expression BeadChip (Illumina, San Diego, CA, USA). Details of subsequent detection and quantitative analyses are available upon request.
7
Gene Expression Profiling of Mouse Tissues
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Total RNA was isolated using an RNeasy Mini Kit (Qiagen) and digested with DNase I (RNase-free DNase, Qiagen) according to the manufacturer’s instructions. Total RNA was amplified, biotinylated, and purified using the Ambion Illumina RNA amplification kit (Ambion) according to the manufacturer’s instructions. Biotinylated cRNA samples (750 ng) were hybridized to each MouseRef-8 v2 Expression BeadChip (Illumina), and signals were detected using Amersham fluorolink streptavidin-Cy3 (GE Healthcare Life Sciences) by following the Illumina bead array protocol. Arrays were scanned with an Illumina BeadArray Reader confocal scanning system according to the manufacturer’s instructions.
Raw data were extracted using the software provided by the manufacturer (Illumina GenomeStudio v2011. 1, Gene Expression Module v1.9. 0). Array data were filtered on the basis of p-values of <0.05 in at least 50% of the samples. The selected probe signal value was logarithmically transformed and normalized using the quantile method. Comparative analyses were carried out using the local pooled error test and fold change. False discovery rate was controlled by adjusting the p-values using the Benjamini–Hochberg algorithm. Hierarchical clustering was performed using average linkage and Pearson distance as a measure of similarity.
Raw data were extracted using the software provided by the manufacturer (Illumina GenomeStudio v2011. 1, Gene Expression Module v1.9. 0). Array data were filtered on the basis of p-values of <0.05 in at least 50% of the samples. The selected probe signal value was logarithmically transformed and normalized using the quantile method. Comparative analyses were carried out using the local pooled error test and fold change. False discovery rate was controlled by adjusting the p-values using the Benjamini–Hochberg algorithm. Hierarchical clustering was performed using average linkage and Pearson distance as a measure of similarity.
8
RNA Isolation and Microarray Analysis of Resected Liver Samples
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Resected liver samples were placed immediately into RNALater (Qiagen, Valencia, CA) on ice. Tissues were homogenized using a Kinematica homogenizer. RNA was isolated from tissues using RNeasy kit (Qiagen). Concentration and quality of RNA were measured by a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies) and by a 2100 Bioanalyzer (Agilent). Gene microarray analysis was run by the UC San Diego Biomedical Genomics Microarray Core Facility using a MouseRef-8 v2 Expression BeadChip (Illumina, San Diego, CA). A principal component analysis (PCA) was conducted on the signals obtained from the data matrix (25,697 probes × 6 samples) with Matlab (Mathworks, Inc., Torrance, CA). Data generated from gene array were analyzed for differential geneexpression. Genes were considered differentially expressed with a p value <0.05 (Mann–Whitney U test), and a Log2 fold change of >1 or < −1. The generated list was analyzed using Ingenuity Pathway Analysis.
9
RNA Quality Analysis and Microarray Profiling
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RNA quality was checked on Bioanalyzer Nanochip (Agilent Technologies). Samples with RNA integrity number (RIN) >7 were subjected to microarray analysis. mRNA transcription profiling was performed on the MouseRef-8 v2 Expression Beadchip (Illumina). The experimental procedure and data analysis were described previously [33 (link)]. Fold change was calculated as the ratio of normalized intensity of treatment group to that of control group. The cutoff for genes to be considered as differentially expressed is set at 1.5 folds.
10
RNA Isolation and Gene Expression Analysis
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RNA was isolated from lung homogenates using the RNeasy mini kit (Qiagen, Venlo, The Netherlands). Biotinylated cRNA was hybridized onto the Illumina MouseRef-8v2 Expression BeadChip and an Illumina iScan array scanner (Eindhoven, The Netherlands) was used to scan samples [16 (link), 36 (link)]. Detailed methods are available in the supplemental material.
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