BALB/c mice were injected subcutaneously (s.c.) with 105 of either CRH5 or EOH6 MM cells. When tumors reached 100 mm3 they were excised and total RNA was extracted. At the same time, RNA was isolated from lungs and kidneys excised from naïve BALB/c mice. RNA expression in the different tissues was evaluated using the Clariom S Mouse Array (Affymetrix). Expression values were normalized and summarized into transcript clusters for analysis using Robust Multi-array Average approach in Array Studio (OmicSoft, Cary, NC). One-way ANOVA was used to look for differential expression between normal and tumor samples, and p-values were adjusted for multiple comparisons using the Benjamini-Hochberg False Discovery Rate (FDR) method. Only candidates with FDR-adjusted p < 0.001 were considered. The data gathered from this analysis were deposited in the Gene Expression Omnibus (GEO) database (Accession Number: GSE122004 ).
Clariom s mouse array
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Clariom S Mouse Array is a comprehensive gene expression microarray designed to analyze the transcriptome of mouse samples. It provides a broad coverage of known and predicted mouse genes and transcripts. The array can be used to measure the expression levels of thousands of mouse genes simultaneously.
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Lab products found in correlation
Genechip fluidics station 450, by Thermo Fisher Scientific (3 mentions)
Expression console, by Thermo Fisher Scientific (3 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Transcriptome analysis console, by Thermo Fisher Scientific (3 mentions)
Wt plus reagent kit, by Thermo Fisher Scientific (3 mentions)
Genechip scanner 3000 7g, by Thermo Fisher Scientific (2 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (2 mentions)
Partek flow, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Bioanalyzer pico chip, by Agilent Technologies (2 mentions)
Thermal fisher 3 ivt pico reagent kit, by Thermo Fisher Scientific (2 mentions)
Rlt buffer, by Qiagen (2 mentions)
3 ivt pico kit, by Thermo Fisher Scientific (1 mentions)
Isolate 2 rna mini kit, by Meridian Bioscience (1 mentions)
Rnase free dnase set, by Qiagen (1 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
Rnaeasy mini kit, by Qiagen (1 mentions)
Gc scanner 3000, by Thermo Fisher Scientific (1 mentions)
Genespring 14, by Agilent Technologies (1 mentions)
19 protocols using clariom s mouse array
1
Transcriptomic Profiling of Mouse Tumor and Normal Tissues
2
Transcriptional Profiling of Apc-Mutant Organoids
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Microarray data used in this study were generated and published before (Smit et al, 2020a (link)) and are publicly available through the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) under accession number GSE143509. In short, to capture the transcriptional profiles of Apc‐mutant organoids on the verge of transformation, Villin‐CreERT2(wild type, WT) and Villin‐CreERT2;Apcfl/fl organoids were plated, and recombination was activated using 1 μM of 4OH‐tamoxifen. After 4 days, organoids were passaged to ensure full recombination, and RNA was isolated 3 days later using the ISOLATE II RNA Mini Kit (BIO‐52073, Bioline). A total of 400 ng of purified RNA was amplified and labeled using the 3′ IVT Pico Kit (Affymetrix) and RNA Amplification Kit (Nugene) according to manufacturer's protocols. Microarray analysis of mouse organoids was performed using Affymetrix Clariom S mouse array by the Dutch Genomics Service and Support Provider (MAD, Science Park, University of Amsterdam, The Netherlands). Washing and staining were performed by the GeneChip Fluidics Station 450, and the scanning was performed using the GeneChip Scanner 3000 7G (both Thermo Fisher Scientific). Data normalization, statistical testing, and extraction of differentially expressed genes were performed using the R2 platform (R2 R2 database, 2019 ).
3
Tumor-Infiltrating CD8+ T Cell Isolation
4
Mouse Gene Expression Profiling Protocol
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RNA was isolated using the RNeasy Mini Kit (Qiagen, Düsseldorf, Germany). Gene expression profiling (GEP) was performed using high-density oligonucleotide arrays (Clariom S mouse, Affymetrix, Santa Clara, CA, USA). Target preparation was performed according to the Affymetrix WTPlus Expression Protocol with approximately 100 ng of total RNA for the analysis on the Affymetrix Clariom S mouse array. Hybridization, washing, and staining of the arrays were done according to the Affymetrix recommendations on the GC Scanner 3000 with G7 update.
To identify differentially expressed genes, statistical analyses were performed in which the experimental samples were compared to the respective baseline samples by using the RMA signal summarization method and ANOVA test implemented in the Affymetrix Transcriptome Analysis Suite (Folder 3_TAC_analysis).
Gene set enrichment analysis (GSEA) was performed with RMA signals calculated with the Affymetrix Expression Console, GSEA software version 4.1, and MSigDB geneset collection v7.4 using default settings and gene set permutation. Pathways showing a false discovery rate (FDR) <0.25 and a normalized enrichment score (NES) > 1.5 were identified.
To identify differentially expressed genes, statistical analyses were performed in which the experimental samples were compared to the respective baseline samples by using the RMA signal summarization method and ANOVA test implemented in the Affymetrix Transcriptome Analysis Suite (Folder 3_TAC_analysis).
Gene set enrichment analysis (GSEA) was performed with RMA signals calculated with the Affymetrix Expression Console, GSEA software version 4.1, and MSigDB geneset collection v7.4 using default settings and gene set permutation. Pathways showing a false discovery rate (FDR) <0.25 and a normalized enrichment score (NES) > 1.5 were identified.
5
Affymetrix microarray analysis of mRNA
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Linear messenger RNA (mRNA) amplification was achieved using the Affymetrix WT-Plus kit (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s guidelines. Briefly, 100 ng of RNA from each sample was used to generate amplified and biotinylated complimentary DNA (cDNA) according to the guidelines of the Affymetrix WT-Plus kit. Target denaturation was performed at 99 °C for 5 min. cDNA was then hybridized to the Clariom S Mouse Array (Affymetrix, Santa Clara, CA, USA) for 16 h at 45 °C while being rotated at 60 rpm. Arrays were then washed and stained using GeneChip Fluidics Station 450 (Affymetrix, CA, USA) and subsequently scanned with Affymetrix Gene Chip Scanner 3000 using Command Console software. Signal space transformation (SST) normalization of raw data was performed using Expression Console software v 1.41. (provided by Affymetrix). GeneSpring 14.9 software (Agilent, Santa Clara, CA, USA) was used to identify differentially expressed transcripts between experimental and control groups. Of note, samples were processed for microarrays in two batches: first, 3 samples for each condition (n = 36) and then 2 samples for each treatment condition (n = 24) for a total of 60 samples representing 5 animals per each ion/dose.
6
Combination Therapy Transcriptome Analysis
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Tumor samples from the Hepa1-6 model collected at the early timepoint or from mice reaching the survival endpoint at day 13 post-randomization (n=21) were processed for transcriptome analysis. Gene expression microarray studies were conducted using the Clariom S Mouse Array (Affymetrix, Santa Clara, CA; GSE153203).
The combination rescue signature was generated by selecting the top differentially expressed genes between tumors from the combination and placebo arms (Bonferroni p<0.05, FC>3 or < 0.33). Genes significantly enriched in the monotherapy groups compared to placebo according to the same criteria were eliminated from the signature to capture only the combination-specific transcriptomic effect.
The combination rescue signature was generated by selecting the top differentially expressed genes between tumors from the combination and placebo arms (Bonferroni p<0.05, FC>3 or < 0.33). Genes significantly enriched in the monotherapy groups compared to placebo according to the same criteria were eliminated from the signature to capture only the combination-specific transcriptomic effect.
7
Liver RNA Isolation and Expression Profiling
8
Transcriptome Profiling of Aging Gastric Organoids
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Total RNA was extracted from young and aged gastric organoids using TRIzol reagent (Invitrogen), and 1 μg of RNA was utilized for Clariom S mouse array (Applied Biosystems). The acquired data were analyzed using gene set enrichment analysis (GSEA) software (Broad Institute).
9
Transcriptome Analysis of Mouse Lung RNA
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Total lung RNA was isolated using PureLink R.N.A. Mini Kit (Invitrogen) according to the manufacture’s instruction and probed using the Clariom S Mouse Array (Applied Biosystems) at the facility (AKESOgen Inc, GA, USA). Raw data that included feature intensity values were converted into summarized expression values using Robust Multi-array Average (RMA), consisting of background correction, quantile normalization, and summarization across all samples. All samples passed QC thresholds for hybridization, labeling, and the expression of housekeeping gene controls (Ake). Principal component analysis was performed for detecting outliers across all chips. Gene expression data generated from Clariom S Mouse Arrays were analyzed. Differentially expressed genes (DEGs) were identified using R Project for Statistical Computing, Partek Flow, and Transcriptome Analysis Console (Applied Biosystems) packages. A false discovery rate (FDR), P value of less than 0.05, was considered significant. The data generated in this publication have been deposited in the NCBI Gene Expression Omnibus and are accessible through GEO Series accession number GSE165969.
10
Transcriptome Analysis of Epididymal Fat in HFD Mice
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Total RNA was extracted from the epididymal fat of WT_A12 and KO_A12 mice fed with a high-fat diet for 12 weeks using the TRI reagent. Then, the obtained RNA was purified using the RNeasy Mini Kit (QIAGEN). Total RNA from three mice per strain was pooled for each array. The transcripts from epididymal fat were measured using a Clariom S Mouse Array (Applied Biosystems). Raw data were normalized with the STT-RMA algorithm using the Applied Biosystems GeneChip Console Software ver.1.3.0. The microarray data have been deposited in the NCBI Gene Expression Omnibus (GEO) (GSE14750).
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