One hundred nanogram of RNA was used for Whole Transcript cDNA synthesis (Affymetrix, inc., Santa Clara, USA). Hybridization, washing and scanning of Affymetrix GeneChip Mouse Gene 1.1 ST arrays was carried out according to standard Affymetrix protocols. All arrays of the small intestine were hybridized in one experiment. Arrays were normalized using the Robust Multi-array Average method58 (link),59 (link). Probe sets were assigned to unique gene identifiers, in this case Entrez IDs. The probes on the Mouse Gene 1.1 ST arrays represent 21,213 Entrez IDs. Array data were analyzed using an in-house, on-line system60 (link).
Genechip mouse gene 1.1 st arrays
Manufactured by Thermo Fisher Scientific
Sourced in Germany
The GeneChip Mouse Gene 1.1 ST arrays are a high-density oligonucleotide microarray platform designed for comprehensive whole-transcript gene expression analysis of mouse samples. The arrays provide comprehensive coverage of the mouse transcriptome, with probes targeting both coding and non-coding transcripts.
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Lab products found in correlation
Whole transcript cdna synthesis, by Thermo Fisher Scientific (4 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Ambion wt expression kit, by Thermo Fisher Scientific (2 mentions)
Nd 1000, by Thermo Fisher Scientific (2 mentions)
Affymetrix genechip wt terminal labeling kit, by Thermo Fisher Scientific (2 mentions)
Ingenuity pathways analysis, by Qiagen (1 mentions)
Genetitan, by Thermo Fisher Scientific (1 mentions)
Rnaeasy kit, by Qiagen (1 mentions)
Ingenuity pathway analysis (ipa), by Qiagen (1 mentions)
Rnalater, by Qiagen (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Wt plus reagent kit, by Thermo Fisher Scientific (1 mentions)
Rneasy microkit columns, by Qiagen (1 mentions)
Agilent 2100, by Agilent Technologies (1 mentions)
Ambion whole transcript expression kit, by Thermo Fisher Scientific (1 mentions)
Genechip wt terminal labeling kit, by Thermo Fisher Scientific (1 mentions)
10 protocols using genechip mouse gene 1.1 st arrays
1
Whole Transcript Profiling of Mouse Small Intestine
2
Microarray Analysis of Intestinal Transcriptome
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One hundred nanogram of RNA was used for Whole Transcript cDNA synthesis (Affymetrix, inc., Santa Clara, USA). Hybridization, washing and scanning of Affymetrix GeneChip Mouse Gene 1.1 ST arrays and Affymetrix GeneChip Porcine Gene 1.1 ST Arrays was carried out according to standard Affymetrix protocols. All arrays of the small intestine were hybridized in one experiment. Arrays were normalized using the Robust Multi-array Average method [23] (link), [24] (link). Probe sets were assigned to unique gene identifiers, in this case Entrez IDs. The probes on the Mouse Gene 1.1 ST arrays represent 21,213 Entrez IDs. The probes on the porcine gene arrays represent 17,118 Entrez IDs [25] . Array data were analyzed using an in-house, on-line system [26] (link). All microarray data have been submitted to the Gene Expression Omnibus (GSE59054).
Both microarray and qPCR techniques have been extensively studied in the past decades and evidence for a strong correlation of the measured gene expression between qPCR and Microarray analysis has been assessed and proven in several papers [27] (link)–[29] . Our own data were in accordance with these studies as comparisons of qPCR data with microarray data of 12 intestinal locations in pigs established that gene expresion patterns were highly similar when using both techniques (data not shown).
Both microarray and qPCR techniques have been extensively studied in the past decades and evidence for a strong correlation of the measured gene expression between qPCR and Microarray analysis has been assessed and proven in several papers [27] (link)–[29] . Our own data were in accordance with these studies as comparisons of qPCR data with microarray data of 12 intestinal locations in pigs established that gene expresion patterns were highly similar when using both techniques (data not shown).
3
Transcriptional Analysis of Murine Jejunum
4
Liver Transcriptomic Profiling of Dietary Interventions
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To reveal the liver transcriptomic profile of the four different diets, whole-genome gene expression was analyzed by microarray analysis. This analysis included seven mock-treated animals from each C, MF, and INT diet groups, and six animals from the CR diet group. One hundred nanograms of RNA was used for Whole Transcript cDNA synthesis (Affymetrix, Santa Clara, CA, USA). Hybridization, washing, and scanning of Affymetrix GeneChip Mouse Gene 1.1 ST arrays were carried out according to standard Affymetrix protocols. Arrays were normalized using the Robust Multiarray Average method [21 (link),22 (link)]. Probe sets were defined according to Dai et al. [23 (link)]. In this method probes are assigned to unique gene identifiers, in this case Entrez IDs. The probes on the Gene 1.1 ST arrays represent 21225 Entrez IDs. For the analysis, only genes having intensity value of >20 on at least five array were taken into account, which resulted in 14 758 genes. Array data have been submitted to the Gene Expression Omnibus, with accession number GSE61233. The hierarchical clustering plot depicting gene expression profile similarity was constructed by using Multiple Experiment Viewer [24 (link)] and the accompanying body weight heatmap was prepared in Excel.
5
Kidney Gene Expression Analysis
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Frozen kidney samples from low single exposures and low combination exposure groups were thawed and total RNA extracted with an RNAeasy kit (Qiagen). RNA samples were sent to the University of North Carolina Functional Genomics Core for global gene expression analysis with GeneChip Mouse Gene 1.1 ST arrays (Affymetrix, Santa Clara). Gene expression data (CEL files) were analyzed using Partek Genomic Suite, v 6.5 (Partek, St. Louis). Data were normalized by robust multiarray analysis and analysis of variance (ANOVA). Differentially expressed genes were identified between classes using the non-parametric rank-product method (P < 0.05), and log2 expression values for each gene were generated. Canonical pathway analysis was generated through Ingenuity Pathway Analysis (Qiagen). Functional classes were compared to a previous report (Desimone et al. 2013 (link)).
6
Transcriptional Profiling of Murine Cecal Tissue
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C57BL/6 mice harboring a reduced altered Schaedler Flora (ASF4; n = 6) or ASF4 mice colonized with M. schaedleri MCS for 10 days (n = 6) were sacrificed. The cecum was washed in phosphate-buffered saline (PBS) to remove contents and stored in RNAlater (Qiagen). RNA was purified from cecal tissue samples using TRIzol (Life Technologies, Inc., Carlsbad, CA, USA) followed by RNeasy Microkit columns (Qiagen, Venlo, the Netherlands). RNA quality was assessed on the Agilent 2100 bioanalyzer (Agilent Technologies, Amsterdam, the Netherlands). RNA was labeled using an Affymetrix WT plus reagent kit and hybridized to GeneChip Mouse Gene 1.1 ST arrays (Affymetrix, Santa Clara, CA). Sample labeling, hybridization to chips, and image scanning were performed according to the manufacturer’s instructions. Microarray analysis was performed using the MADMAX pipeline (92 (link)). Quality control was performed, and all arrays met our criteria. A custom annotation that combines all individual probes for a gene (93 (link)) was used. Expression values were calculated and normalized using the robust multichip average (RMA) method (94 (link)), and significant differences were assessed using the paired intensity-based moderated T statistic (IBMT) (95 (link)). Pathway analysis was performed by gene set enrichment analysis (96 (link)); upstream regulator analysis (Ingenuity) was also performed.
7
Terminal Ileum RNA Isolation and Microarray Analysis
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A piece of terminal ileum from each mouse was snap frozen in liquid nitrogen and stored afterward at −80°C. RNA was isolated with the RNeasy kit (Qiagen, Valencia, CA, USA). Quantity of RNA was measured with the ND-1000 (NanoDrop Technologies, Thermo Fisher Scientific, Breda, the Netherlands) and quality of RNA was assessed with the Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). Total RNA (100 ng) was labeled utilizing the Ambion WT Expression kit (Life Technologies Ltd., Bleiswijk, the Netherlands) and the Affymetrix GeneChip WT Terminal Labeling kit (Affymetrix, Santa Clara, CA, USA). After labeling, samples were hybridized to Affymetrix GeneChip Mouse Gene 1.1 ST arrays. An Affymetrix GeneTitan Instrument was used for hybridization, washing, and scanning of the array plates. Bioconductor packages integrated in an online pipeline were used for quality control of the data (12 (link), 13 (link)). Probe sets were redefined using current genome information (14 (link)). Probes were reorganized based on the Entrez Gene database (remapped CDF v14.1.1). Robust Multi-array Analysis preprocessing algorithm available in the Bioconductor library affyPLM (15 (link)) was used to obtain normalized expression estimates from the raw intensity values.
8
Mouse Whole Transcriptome Expression Profiling
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Per offspring, colon and SI samples were analysed as described previously [43 (link)], and in total, six male and six female samples were used in this analysis (three of the nine females were not included in the microarray analysis due to budget limitations). In brief, 100 ng of purified RNA was used for the preparation of labelled cDNA, applying the Ambion Whole Transcript (WT) Expression Kit (Life Technologies, Carlsbad, CA, USA) in combination with the Affymetrix GeneChip WT Terminal Labeling Kit (Affymetrix, Santa Clara, CA, USA). All samples were hybridized at one time point to Affymetrix GeneChip Mouse Gene 1.1 ST arrays according to standard Affymetrix protocols. Quality control and normalization were performed using Bioconductor software packages integrated in an on-line pipeline [44 (link)]. Normalized expression estimates of probe sets were computed by the robust multiarray (RMA) analysis algorithm available in the Bioconductor library AffyPLM using default settings [45 (link)]. Probe sets were redefined according to Dai et al. [46 (link)] and assigned to unique gene identifiers (IDs) of the Entrez Gene database, resulting in 21,187 assigned Entrez IDs. Array data were submitted to the Gene Expression Omnibus and are available under accession number GSE57516.
9
Whole Transcript Analysis of Mouse Intestine
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The microarray was performed as described previously23 (link). Briefly, One hundred nanogram of RNA was used for Whole Transcript cDNA synthesis (Affymetrix, inc., Santa Clara, USA). Hybridization, washing and scanning of Affymetrix GeneChip Mouse Gene 1.1 ST arrays was carried out according to standard Affymetrix protocols. All arrays of the small intestine were hybridized in one experiment. Arrays were normalized using the Robust Multiarray Average method24 (link),25 (link). Probe sets were assigned to the unique gene identifiers Entrez IDs. The probes on the Mouse Gene 1.1 ST arrays represent 21,213 Entrez IDs. Array data were analyzed using an in-house, on-line system26 (link).
10
Transcriptomic Analysis of Ileal Tissue
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A part of the terminal ileum from each mouse was snap frozen in liquid nitrogen and stored at −80°C. RNA was isolated with the RNeasy kit (Qiagen, Valencia, CA, USA). Quantity of RNA was measured with the ND-1000 (NanoDrop Technologies, Thermo Fisher Scientific, Breda, the Netherlands) and quality of RNA was assessed with the Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). Total RNA (100 ng) was labeled utilizing the Ambion WT Expression kit (Life Technologies Ltd., Bleiswijk, the Netherlands) and the Affymetrix GeneChip WT Terminal Labeling kit (Affymetrix, Santa Clara, CA, USA). After labeling, samples were hybridized to Affymetrix GeneChip Mouse Gene 1.1 ST arrays. An Affymetrix GeneTitan Instrument was used for hybridization, washing, and scanning of the array plates. Bioconductor packages integrated in an online pipeline were used for quality control of the data (17 (link), 18 (link)). Probe sets were redefined using current genome information (19 (link)). Probes were reorganized based on the Entrez Gene database (remapped CDF v14.1.1). Robust Multiarray Analysis preprocessing algorithm available in the Bioconductor library affyPLM (20 (link)) was used to obtain normalized expression estimates from the raw intensity values.
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