For total RNA isolation, we followed the instruction of the Ambion Purelink RNA mini kit. cDNA was synthesized from RNA template by using SuperScript III reverse transcriptase (Invitrogen). Real-time PCR amplifications were performed in 96-well optical reaction plates with SYBR Green by using Taq DNA polymerase (Qiagen). We used Affymetrix Gene 1.0-ST microarrays for gene expression analysis of mouse liver RNAs. Microarray data were analyzed using the Bioconductor package “ArrayTools”. Differentially expressed genes were selected by using a cutoff of P <= 0.005 and fold change >= 1.5. Heat maps were created by using Cluster v3.0 and visualized by Java Treeview. Raw data was deposited into the GEO database (accession number GSE73911).
Gene 1.0 st microarrays
Manufactured by Thermo Fisher Scientific
The Gene 1.0 ST microarrays are a gene expression profiling platform designed for whole-transcript coverage analysis. The microarrays contain probes targeting known and predicted genes to provide a comprehensive view of the transcriptome.
Automatically generated - may contain errors
Lab products found in correlation
Taq dna polymerase, by Qiagen (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
Ambion wt expression kit, by Thermo Fisher Scientific (1 mentions)
Affymetrix genechip wt terminal labeling kit, by Thermo Fisher Scientific (1 mentions)
Genechip scanner, by Thermo Fisher Scientific (1 mentions)
U133plus2.0 microarray, by Thermo Fisher Scientific (1 mentions)
U133a microarray, by Thermo Fisher Scientific (1 mentions)
3 protocols using gene 1.0 st microarrays
1
Mouse Liver Transcriptome Analysis
2
Microarray RNA Expression Profiling
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Total RNA (200 ng) was converted to sense strand cDNA, amplified using the Ambion WT Expression kit (Life Technologies Cat. #4440536), and labeled with Affymetrix GeneChip WT terminal labeling kit (Affymetrix Cat. #900671), (described in more detail in Additional file 1 ). The labeled cDNA was hybridized to Gene 1.0 ST microarrays (Affymetrix Cat. #901085) and analyzed on an Affymetrix GeneChip Scanner. Individual CEL files for each of the patient samples were normalized using the standard Affymetrix Gene 1.0 ST CDF and RMA [14 (link)].
3
Microarray Analysis of Adipose Tissue
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Microarray analysis of paired subcutaneous and omental adipose tissue samples and adipose tissue samples from the SibPair parents study was performed using U133 plus 2.0 microarray (Affymetrix, Santa Clara, CA). Microarray analysis of adipose tissue from the VLCD study was performed using U133A microarray (Affymetrix), and perirenal adipose tissue was analyzed using Gene 1.0 ST microarrays (Affymetrix). All microarray analysis was performed according to the manufacturer's instructions (Affymetrix), and expression data were analyzed using the Robust Multi‐array Average algorithm. SLC19A3 expression was assessed using probe sets 220736_at and 8059538 for U133 microarrays and Gene 1.0 ST microarrays, respectively.
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