A 100ng aliquot of total RNA was linearly amplified. Then, 5.5µg of cDNA was labeled and fragmented using the GeneChip WT PLUS reagent kit (Affymetrix Santa Clara, CA) following the manufacturer's instructions. Labeled cDNA targets were hybridized to Affymetrix GeneChip Mouse Gene ST 2.0 arrays for 16 h at 45°C rotating at 60rpm. The arrays were washed and stained using a Fluidics Station 450 and scanned using a GeneChip Scanner 3000. Signal intensities were quantified by Affymetrix Expression Console version 1.3.1. Background correction and quantile normalization were performed to adjust for technical bias, and probe-set expression levels were calculated by the RMA method. After filtering above noise cutoff, there are 9,528 probe-sets that were tested by linear model. A variance smoothing method with fully moderated t-statistic was employed for this study and was adjusted by controlling the mean number of false positives. With a combined cutoff of 2-fold change and p-value of 0.0001 (controlling 1 false positive over all probe-sets), we declared 500 probe-sets as differential gene expression between KO and WT preadipocytes. GEO file: ‘QS wild type and Aldh1a1 KO preadipocytes 2015’.
Expression console version 1
Manufactured by Thermo Fisher Scientific
The Expression Console version 1.1 is a software tool designed for the analysis and visualization of gene expression data. It provides users with a platform to import, manage, and analyze data from various gene expression experiments.
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Lab products found in correlation
Agilent 2100 bioanalyzer, by Agilent Technologies (3 mentions)
Genechip mouse gene st 2.0 arrays, by Thermo Fisher Scientific (2 mentions)
Genechip wt plus reagent kit, by Thermo Fisher Scientific (2 mentions)
Genechip human gene 1.0 st array, by Thermo Fisher Scientific (2 mentions)
Ambion wt expression kit, by Thermo Fisher Scientific (2 mentions)
Genechip system, by Thermo Fisher Scientific (2 mentions)
Scanner 3000 7g workstation fluidics 450, by Thermo Fisher Scientific (2 mentions)
Rna 6000 nano kit, by Agilent Technologies (2 mentions)
Gene 1.0 st array, by Thermo Fisher Scientific (1 mentions)
Genechip human exon 1.0 st array, by Thermo Fisher Scientific (1 mentions)
Hybridization oven 645, by Thermo Fisher Scientific (1 mentions)
Affymetrix genechip wt terminal labeling kit, by Thermo Fisher Scientific (1 mentions)
Human gene 1.1 st array, by Thermo Fisher Scientific (1 mentions)
Affymetrix genechip human gene 1.0 st array, by Thermo Fisher Scientific (1 mentions)
Mouse gene 1.0 st array, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Trizol solution, by Thermo Fisher Scientific (1 mentions)
Genespring gx software version 12, by Agilent Technologies (1 mentions)
High pure rna isolation kit, by Roche (1 mentions)
14 protocols using expression console version 1
1
Transcriptomic Profiling of Preadipocytes
2
Transcriptomic Analysis of Aldh1a1 KO Preadipocytes
3
Transcriptome Analysis of BJ, BJEL, and BJELM Cells
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Transcriptome analysis was performed using an Affymetrix Gene 1.0 ST Array in two biological replicates for each cell line, providing 1 μg of extracted RNA for library production. For comparing BJ, BJEL, and BJELM cells’ generated transcriptomes, we normalized all raw CEL files with the Affymetrix software Expression Console version 1.3.1 to calculate probe-set signal intensities using RMA algorithms with default settings. High reproducibility between the corresponding biological replicates was evaluated by calculating the Pearson correlation coefficient and skewness parameter between replicates and between BJEL and BJELM relative to BJ (Additional file 2 : Figure S2).
To identify differentially expressed genes (DEGs), we compared BJEL versus BJ and BJELM versus BJ (in biological replicates). Thus, to identify confident DEGs, we used a modified t-test [6 ] for measurements coming from independent normal populations with unequal variances; this method aims to specifically address the question of differential expression in tests involving two samples (BJ versus BJEL or BJ versus BJELM) in which the experiments were performed in repeats. Finally, the probability of having a t-statistic value by chance was calculated and a threshold (significance level of 0.05) was applied.
To identify differentially expressed genes (DEGs), we compared BJEL versus BJ and BJELM versus BJ (in biological replicates). Thus, to identify confident DEGs, we used a modified t-test [6 ] for measurements coming from independent normal populations with unequal variances; this method aims to specifically address the question of differential expression in tests involving two samples (BJ versus BJEL or BJ versus BJELM) in which the experiments were performed in repeats. Finally, the probability of having a t-statistic value by chance was calculated and a threshold (significance level of 0.05) was applied.
4
Profiling Human Medulloblastomas via Microarray
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Human primary medulloblastomas were profiled on Affymetrix Genechip Human Exon 1.0ST arrays at The Centre for Applied Genomics in Toronto, Canada (www.tcag.ca ). Expression analysis was performed using Affymetrix Expression Console (Version 1.1) as previously described [26 (link)]. Additional, publically available medulloblastoma expression data sets were obtained from NCBI Gene Expression Omnibus and used to validate our findings [9 (link),27 (link)]. Subgrouping of tumors was performed using an 84-gene expression classifier [28 (link)].
5
Microarray Gene Expression Analysis Protocol
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In order to evaluate the integrity of the RNA, a microfluidic analysis was performed using an Agilent 2100 Bioanalyzer with the RNA6000 nano kit (Agilent Technologies, Palo Alto, CA, USA). For the microarray analysis, we used only RNA samples whose RNA integrity number (RIN) was greater than 8.5. The gene expression was analyzed using a GeneChip® Human Gene 1.0 ST Array (Affymetrix, Santa Clara, CA, USA) containing 764,885 probes (and supporting 28,869 genes). Target cDNA was prepared from 200 ng of total RNA with the Ambion® WT Expression kit (Ambion, Austin, TX, USA) and the Affymetrix® GeneChip® WT Terminal Labeling kit (Affymetrix). Hybridization to the microarrays, washing, staining and scanning were performed using the GeneChip® system (Affymetrix) composed of a Scanner 3000 7G Workstation Fluidics 450 and a Hybridization Oven 645. The scanned image data were processed using the Affymetrix® Expression Console™ Version 1.1. The fold-change for each gene was evaluated using a Gene Expression Analysis with the Partek® Genomics Suite 6.5 software program (Partech, Münster, Germany). Genes with an expression level greater than 2-fold or less than 0.5 were recognized as being significantly different.
6
Transcriptional Profiling of Medulloblastoma
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Transcriptional profiling of BMI1kd versus wild-type MB cell lines (DAOY) on Affymetrix Gene Chip Genome 133 2.0 Plus Expression arrays were downloaded from Gene Expression Omnibus (GSE7578). Similarly, human primary MB expression data across a 285 tumours previously profiled on Affymetrix Human Gene 1.1ST arrays were downloaded from GSE37382. All CEL files were analysed using Affymetrix Expression Console (Version 1.1) as previously described in Northcott et al. [3 (link)]. Genome-wide statistically significant differences in gene expression patterns were calculated using the Wilcoxon Rank Sum Test with Benjamini-Hochberg FDR correction (q < 0.01) in MultiExperiment Viewer (MeV). Statistically significant gene sets were further filtered on the basis of absolute fold-changes greater or equal to 1.5. Pathway analysis was performed using GSEA Molecular Signature Database (MSigDB) using the curated pathways described, and an FDR q-value below 0.05. Unsupervised hierarchical clustering of BMI1-high, TP53-low versus BMI1-low, TP53-low Group 4 medulloblastomas was performed using the top 1500 genes with the highest standard deviation using the Pearson Correlation metric and bootstrapping as described previously [3 (link)].
7
Arabidopsis Transcriptome Analysis by Microarray
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Briefly, microarrays were processed by the W. M. Keck Center for Comparative and Functional Genomics in the Roy J. Carver Biotechnology Center at the University of Illinois at Urbana-Champaign. Affymetrix GeneChip® 3′ Expression Arabidopsis ATH1 Genome Array was hybridized with antisense RNA prepared from 100 ng of total RNA and scanned with a GeneChip® Scanner model 3000 7G Plus. From the image files, fluorescence intensity (CEL) files were generated and analyzed with Affymetrix Expression Console version 1.1. Raw data was processed with the GCRMA algorithm and clustered using Weighted Gene Co-expression Network Analysis 8,9 (WGCNA). For selected clustered modules, over-representation analyses of Gene Ontology terms and KEGG pathways were performed.
8
Adipocyte Differentiation Gene Expression
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To determine whether the hub genes were differentially expressed during in vitro differentiation of primary human adipocytes, we analyzed gene expression profiles of subcutaneous adipose tissue obtained from healthy subjects undergoing cosmetic liposuction (n = 12)41 (link). The gene expression dataset was publicly accessible via GEO (accession number: GSE25910). Preadipocytes were isolated from the adipose tissue and in vitro differentiated to adipocytes. The cells were lysed at day 4/5 (early), 8 (middle) and 12 (late) of differentiation. From the samples, biotinylated complementary RNA was prepared and hybridized to Affymetrix GeneChip Human Gene 1.0 ST Array (Affymetrix Inc., Santa Clara, CA). Pre-processing and quality control was performed using the Affymetrix Expression Console version 1.1 by summarization, background correction and normalization41 (link). Differential expression analysis across three time points was the same with GSE35411 (Experiment 2).
9
Transcriptomic Analysis of Mgat2-Deficient T Cells
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CD4+ T cells isolated from Mgat2f/f mice and L-PHA-CD4+ T cells isolated from Mgat2f/f::Lck-Cre+ mice were used for analysis. The RNeasy mini kit (Qiagen, Valencia, CA) was used for RNA extraction. Gene expression was assessed using the Affymetrix Mouse Gene 1.0 ST arrays in triplicate. Array data were quantified with Expression Console version 1.1 software (Affymetrix, Santa Clara, CA) using the PLIER Algorithm default values. Expression values were then filtered as present/absent at expression 100. The Cyber-T web server was used for data analysis and to compare samples.
10
Transcriptome Analysis of Genotype Differences
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