Microarray analysis was performed on liver samples from the mice fed the three different diets as well as on Hepa1‐6 hepatoma and differentiated 3T3‐L1 adipocyte cells incubated with different fatty acids. RNA was purified with RNeasy Minikit columns (Qiagen) and analyzed for quality with RNA 6000 Nano chips on the Agilent 2100 bioanalyzer (Agilent Technologies, Amsterdam, The Netherlands). One microgram of RNA was used for cDNA synthesis using the First Strand cDNA synthesis kit (Thermo Scientific). Purified RNA (100 ng) was labeled with the Ambion WT expression kit (Invitrogen) and hybridized to an Affymetrix Mouse Gene 1.1 ST array plate (mouse liver, Hepa1‐6 cells) or 2.1 ST array plate (3T3‐L1 adipocytes) (Affymetrix, Santa Clara, CA). Hybridization, washing, and scanning were carried out on an Affymetrix GeneTitan platform. Scans of the Affymetrix arrays were processed using packages from the Bioconductor project. Arrays were normalized using the robust multi‐array average method.46 , 47 Probe sets were defined by assigning probes to unique gene identifiers, for example, Entrez ID.48 For the Hepa1‐6 and 3T3‐L1 cells, the total gene set (24 973 probe sets) was filtered to only include genes with mean signal >20, yielding 10 379 and 11 504 genes, respectively. Microarray data were submitted to the Gene Expression Omnibus (accession number pending).
Mouse gene 1.1 st array plate
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Mouse Gene 1.1 ST array plate is a microarray designed for the analysis of gene expression in mouse samples. It contains probes targeting mouse genes and transcripts, allowing for the measurement of gene expression levels in mouse biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Ambion wt expression kit, by Thermo Fisher Scientific (5 mentions)
Genetitan platform, by Thermo Fisher Scientific (4 mentions)
Rneasy mini kit column, by Qiagen (3 mentions)
Rna 6000 nano chip, by Agilent Technologies (3 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (3 mentions)
First strand cdna synthesis kit, by Thermo Fisher Scientific (3 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Rneasy plus kit, by Qiagen (1 mentions)
Genetitan instrument, by Thermo Fisher Scientific (1 mentions)
Genechip wt terminal labeling and hybridization kit, by Thermo Fisher Scientific (1 mentions)
6 protocols using mouse gene 1.1 st array plate
1
Microarray analysis of fatty acid effects
2
Transcriptomic Profiling of Fatty Acid Effects
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Microarray analysis was performed on Hepa1-6 hepatoma cells incubated with different fatty acids. RNA was purified with RNeasy Minikit columns (Qiagen) and analyzed for quality with RNA 6000 Nano chips on the Agilent 2100 bioanalyzer (Agilent Technologies, Amsterdam, The Netherlands). One microgram of RNA was used for cDNA synthesis using the First Strand cDNA synthesis kit (Thermo Scientific). Purified RNA (100 ng) was labeled with the Ambion WT expression kit (Invitrogen) and hybridized to an Affymetrix Mouse Gene 1.1 ST array plate (Affymetrix, Santa Clara, CA). Hybridization, washing, and scanning were carried out on an Affymetrix GeneTitan platform. Scans of the Affymetrix arrays were processed using packages from the Bioconductor project. Arrays were normalized using the robust multi-array average method [27 (link),28 (link)]. Probe sets were defined by assigning probes to unique gene identifiers, e.g., Entrez ID [29 (link)]. The total gene set (24,973 probe sets) was filtered to only include genes with mean signal > 20, yielding 10,379 genes. Microarray data were submitted to the Gene Expression Omnibus (accession number pending).
3
Affymetrix Mouse Gene 1.1 ST Array Analysis
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Total RNA was harvested from the cells using an RNeasy Plus kit (Qiagen, Crawley, UK), according to the manufacturer's instructions. RNA was quantified and quality-controlled using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) to determine RNA purity and integrity. Replicate 250-ng samples of total RNA, derived from two separate wells/time-point, were first processed using the Ambion WT Expression Kit (Life Technologies, Carlsbad, CA, USA) to generate amplified and biotinylated sense-strand DNA targets from the entire genome without bias. Sense-strand DNA samples were then labeled and hybridized to the Affymetrix Mouse Gene 1.1 ST Array Plate using the GeneChip WT terminal labeling and hybridization kit (Affymetrix, Santa Clara, CA, USA), according to the manufacturer's recommendation. Individual arrays interrogate >28,000 annotated transcripts using >770,000 distinct probes. Hybridization, washing, and scanning of the 64 arrays were performed in a single run using the Affymetrix GeneTitan instrument, according to the manufacturer's recommendations.
4
Transcriptome Analysis of Pregnant Mouse Colon
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RNA was purified from the proximal colon of mice (n = 5 per group) and RNA expression profiling was performed with an Affymetrix Mouse Gene 1.1 ST array plate (Affymetrix, Santa Clara, CA, USA) as previously described26 (link). Only probe sets with a fold-change of at least 1.2 (up/down) and a p-value < 0.05 were considered to be significantly different.
To gain insight into the biological role of the differently expressed genes during pregnancy, we investigated the pathways in which these genes are involved using Ingenuity Pathway Analysis (IPA) (Ingenuity System). Our IPA analyses included comparison of differentially regulated genes in the colon of pregnant and non-pregnant B6 and BALB/c mice.
To gain insight into the biological role of the differently expressed genes during pregnancy, we investigated the pathways in which these genes are involved using Ingenuity Pathway Analysis (IPA) (Ingenuity System). Our IPA analyses included comparison of differentially regulated genes in the colon of pregnant and non-pregnant B6 and BALB/c mice.
5
Microarray Expression Analysis Protocol
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Purified RNA was labeled with the Ambion WT Expression Kit (Carlsbad) and hybridized to an Affymetrix Mouse Gene 1.1 ST array plate (Affymetrix, Santa Clara, CA). Hybridization, washing, and scanning were carried out on an Affymetrix GeneTitan platform according to the manufacturer's instructions. Normalized expression estimates were obtained from the raw intensity values applying the robust multiarray analysis preprocessing algorithm available in the Bioconductor library affyPLM with default settings (Bolstad, Irizarry, Astrand, & Speed, 2003 ; Irizarry et al., 2003 ). Probe sets were defined according to Dai et al. (2005 ). In this method, probes are assigned to Entrez IDs as a unique gene identifier. In this study, probes were reorganized based on the Entrez Gene database, build 37, Version 1 (remapped CDF v22). q values were calculated using an intensity‐based moderated T‐statistic (Sartor et al., 2006 ). Genes were defined as significantly changed when q < .05.
6
Microarray Analysis of Fatty Acid Effects
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Microarray analysis was performed on Hepa1-6 hepatoma cells incubated with different fatty acids. RNA was purified with RNeasy Minikit columns (Qiagen) and analysed for quality with RNA 6000 Nano chips on the Agilent 2100 bioanalyzer (Agilent Technologies, Amsterdam, The Netherlands). One microgram of RNA was used for cDNA synthesis using the First Strand cDNA synthesis kit (Thermo Scientific). Purified RNA (100 ng) was labeled with the Ambion WT expression kit (Invitrogen) and hybridized to an Affymetrix Mouse Gene 1.1 ST array plate (Affymetrix, Santa Clara, CA). Hybridization, washing, and scanning were carried out on an Affymetrix GeneTitan platform. Scans of the Affymetrix arrays were processed using packages from the Bioconductor project. Arrays were normalized using the robust multi-array average method (4, 22) . Probe sets were defined by assigning probes to unique gene identifiers, e.g., Entrez ID (10) . The total gene set (24,973 probe sets) was filtered to only include genes with mean signal > 20, yielding 10,379 genes. Microarray data were submitted to the Gene Expression Omnibus (accession number pending).
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