Total RNA was extracted using mini RNeasy kit (Qiagen). cDNAs were obtained from 100 ng of total RNA per sample (GeneChip cDNA synthesis Kit; Affymetrix) and hybridized to Mogene (Affymetrix “MoGene 1.0 ST”) by DNA Microarray Core Facility (UCI). Four (PFC) and three (vStr) microarrays/genotype were used. Normalization and analysis were performed using Partek Genomics Suite software, Genecodis software and DAVID (See SM ).
Mogene 1.0 st
Manufactured by Thermo Fisher Scientific
The MoGene 1.0 ST is a laboratory equipment product from Thermo Fisher Scientific. It is designed for gene expression analysis. The device performs tasks related to the measurement and quantification of gene expression levels.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (3 mentions)
Genechip cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Mta 1, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Wt expression kit, by Thermo Fisher Scientific (1 mentions)
Gene expression console software, by Thermo Fisher Scientific (1 mentions)
4 protocols using mogene 1.0 st
1
Transcriptome analysis of brain regions
2
Transcriptome analysis of brain regions
3
Microarray Analysis of Tissue Samples
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Total RNA was isolated using Trizol (Life Technologies) and purified using the RNeasy Mini Kit (QIAGEN). It was then assessed for degradation using an Advanced Analytical Agilent Fragment Analyzer. For each condition, the 3 best quality samples were selected for liver and muscle and 4 for heart and taken further for microarray analysis. Microarray analysis was performed using the Affymetrix MoGene 1.0ST for liver and quadriceps and the similar but slightly enhanced Affymetrix MTA 1.0 (an array with probes that target splice junctions that is also known as Clariom D) for heart. Microarray data were normalized using the rma function from the R Oligo package (Carvalho and Irizarry, 2010 (link)). Differential expression was performed using the limma package (Smyth, 2005 ).
4
Affymetrix Microarray Analysis of Mouse Gene Expression
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Equal amounts of RNA from five mice per group were pooled within each group. Microarrays were performed as previously described57 (link). Mouse gene ST microarray chips were used for hybridization (MoGene 1.0 ST, Affymetrix). The WT expression kit (Ambion) was used for complementary RNA preparation from the total RNA. The hybridization, wash and scan were done according to the Affymetrix kits and procedures specific to the mouse gene ST chips. After the scan, the quality controls of the hybridization were checked using the Affymetrix Gene Expression Console software. Using the Affymetrix APT suite tools, we normalized the data by the RMA-Sketch procedure and computed the signal detection P values using the DABG algorithm. All the probe sets that have the DABG P value >0.05 in all conditions were discarded from the analysis. The rest of probes sets were kept for fold-change analysis. Functional annotation and pathway analysis was done using the DAVID web tool58 (link). Both tools were fed with the list of selected official gene names as input, and the threshold of significance was set by default to P values <0.05.
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