Gene expression profiles were investigated using a high-density oligonucleotide probe array [GeneChip®, Human Clariom S Array (Affymetrix)] as in our previous report (Ueta et al., 2020 (link); Ueta et al., 2021a (link)). Total RNA was extracted with the QIAGEN RNeasy kit (Qiagen, Valencia, CA). We used approximately 337,100 probe sets covering more than 20,800 genes. We followed Affymetrix instructions throughout. Scanned microarray images were obtained on a 3000 7G GeneChip Scanner (Affymetrix) using the default settings. Images were visually inspected to detect hybridization artifacts.
Genechip human clariom s array
Manufactured by Thermo Fisher Scientific
Sourced in United States
The GeneChip Human Clariom S Array is a high-density microarray designed for gene expression analysis. It provides comprehensive coverage of the human transcriptome, enabling the detection and quantification of both coding and non-coding RNA transcripts.
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Lab products found in correlation
Genechip wt plus reagent kit, by Thermo Fisher Scientific (6 mentions)
Genechip fluidics station 450, by Thermo Fisher Scientific (5 mentions)
Genechip scanner 3000 7g, by Thermo Fisher Scientific (3 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Genechip scanner 3000, by Thermo Fisher Scientific (2 mentions)
Transcriptome analysis console software, by Thermo Fisher Scientific (2 mentions)
Rneasy kit, by Qiagen (1 mentions)
Genechip wt amplification kit, by Thermo Fisher Scientific (1 mentions)
Genechip command console software, by Thermo Fisher Scientific (1 mentions)
Genechip wt terminal labeling kit, by Thermo Fisher Scientific (1 mentions)
Gcs3000 scanner, by Thermo Fisher Scientific (1 mentions)
Microarray suite software, by Thermo Fisher Scientific (1 mentions)
Rna bioanalyzer, by Agilent Technologies (1 mentions)
Wtplus kit, by Thermo Fisher Scientific (1 mentions)
Bioanalyzer rna 6000 nano kit, by Agilent Technologies (1 mentions)
Expression console software 1, by Thermo Fisher Scientific (1 mentions)
Agilent 4200 tapestation, by Agilent Technologies (1 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid protein assay kit, by Merck Group (1 mentions)
Protease and phosphatase inhibitor cocktail, by Merck Group (1 mentions)
12 protocols using genechip human clariom s array
1
High-Throughput Gene Expression Profiling
2
Transcriptomic Analysis of RNA Samples
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Total RNA samples were sent to Macrogen (Seoul, Korea) for assessment using the Clariom™ S Assay, Human. The ND-2000 spectrophotometer (NanoDrop, Wilmington, NC, USA) was used to detect RNA purity, and the Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) was used to detect RNA integrity.
For the Affymetrix whole-transcript (WT) expression array process, the GeneChip WT PLUS Reagent Kit (Affymetrix, Santa Clara, CA, USA) was used according to the manufacturer’s instructions. cDNA was synthesized using the GeneChip WT Amplification Kit (Affymetrix) according to the manufacturer’s instructions.
Thereafter, sense cDNA was fragmented and labeled with biotin using terminal deoxynucleotidyl transferase using the GeneChip WT Terminal Labeling Kit (Affymetrix). Approximately 5.5 μg of labeled DNA target was incubated at 45 °C for hybridization with the Affymetrix GeneChip Human Clariom S Array for 16 h. After washing and staining on the GeneChip Fluidics Station 450, the hybridized arrays were scanned on the GCS3000 Scanner (Affymetrix). The Affymetrix® GeneChip™ Command Console software was used to calculate the signal values.
Gene enrichment, pathway, and functional annotation analyses were performed to obtain a probe list using the DAVID functional annotation tool [30 (link),31 (link)].
For the Affymetrix whole-transcript (WT) expression array process, the GeneChip WT PLUS Reagent Kit (Affymetrix, Santa Clara, CA, USA) was used according to the manufacturer’s instructions. cDNA was synthesized using the GeneChip WT Amplification Kit (Affymetrix) according to the manufacturer’s instructions.
Thereafter, sense cDNA was fragmented and labeled with biotin using terminal deoxynucleotidyl transferase using the GeneChip WT Terminal Labeling Kit (Affymetrix). Approximately 5.5 μg of labeled DNA target was incubated at 45 °C for hybridization with the Affymetrix GeneChip Human Clariom S Array for 16 h. After washing and staining on the GeneChip Fluidics Station 450, the hybridized arrays were scanned on the GCS3000 Scanner (Affymetrix). The Affymetrix® GeneChip™ Command Console software was used to calculate the signal values.
Gene enrichment, pathway, and functional annotation analyses were performed to obtain a probe list using the DAVID functional annotation tool [30 (link),31 (link)].
3
Transcriptional Profiling of OCT4 and NRF1 in Prostate Cancer
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To characterize the transcriptional program regulated by OCT4 and NRF1, we performed microarray analysis of PC cells (LNCaP, 22Rv1, DU145-CR, parental DU145, and PC3-CR after 72 h of being transfected with siControl, and two siRNAs targeting OCT4 or NRF1. RNA samples were validated to be high quality (RNA integrity score >9.0) by using RNA bioanalyzer (Agilent, Waldbronn, Germany). For gene expression microarrays, the GeneChip Human Clariom S Array (Affymetrix, Santa Clara, CA) was used in accordance with the manufacturer’s protocol. Data analysis was performed using the Affymetrix Microarray Suite software. To compare arrays, normalization was performed on data from all the probe sets. Kyoto Encyclpedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID 6.7, http://david.ncifcrf.gov/ ).
4
Affymetrix GeneChip Clariom S Array Protocol
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RNA was converted to cDNA and biotin labelled using the WT plus kit from Affymetrix before being applied to the Affymetrix GeneChip® Human Clariom S Array (Affymetrix, Sunnyvale, CA, USA) with one array being used for each RNA sample. Following hybridization, the arrays were washed and stained on the GeneChip® Fluidics Station 450 (Affymetrix, Sunnyvale, CA, USA) using the appropriate fluidics script, before being inserted into the Affymetrix autoloader carousel and scanned using the GeneChip® Scanner 3000 (Affymetrix, Sunnyvale, CA, USA). All scanned array images were visually inspected for chip surface artefacts that could adversely impact the data.
5
miR-195 Regulation in Macrophages
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RNA from miR-195-THP1 macrophages and SCR-THP-1 macrophages was isolated as described above and RNA quality was assessed using Bioanalyzer RNA 6000 Nano Kit (Agilent). Gene expression was determined using GeneChip Human Clariom S arrays (Affymetrix). Results were analysed using Transcriptome Analysis Console (TAC) Software (Affymetrix). Ingenuity Pathway Analysis (IPA; https://www.qiagenbioinformatics.com/products/ingenuitypathway-analysis ) was performed to identify the pathways and biological functions associated to miR-195 downregulated genes.
6
Gene Expression Profiling of Colon Tissues
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RNA was extracted from isolated normal, adenomatous and carcinomatous glands, and RNA quality was assessed using the Agilent 2100 Bioanalyzer (Agilent). All RNA used was confirmed to be of good quality. In addition, we used 20 ng of RNA for each array. This array measured 21 453 mRNA transcripts. Probe labeling, chip hybridization, and scanning were performed according to the manufacturer's instructions. Gene expression was determined using GeneChip Human Clariom S arrays (Affymetrix) and Transcriptome Analysis Console software (Affymetrix).
7
Transcriptome Analysis of Nutrient Intake
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Microarray analysis was performed on samples taken at 0 and 120 min after the intake of F. Isolated RNA samples were sent to Takara Bio Inc. (Kusatsu, Japan). Total RNA yield was quantified using a Nanodrop 2000 spectrophotometer (Nanodrop Technologies, Wilmington, DE), and integrity was measured using the Agilent 4200 TapeStation (Agilent Technologies, Palo Alto, CA).
An equal amount (200 ng) of RNA prepared from the participants was mixed. Biotinylated cDNA was prepared from 100 ng of total RNA using the GeneChip WT PLUS Reagent Kit (Thermo Fisher Scientific, Waltham, MA) following the manufacturer’s instructions. Following fragmentation, 2 μg of single-stranded cDNA was hybridized for 16 h at 45°C on GeneChip Clariom S Array human (Thermo Fisher Scientific). Arrays were washed and stained in the GeneChip Fluidics Station 450 (Thermo Fisher Scientific). Clariom S array was scanned using the GeneChip Scanner 3000 7G (Thermo Fisher Scientific). Data were analyzed using the Expression Console Software 1.4 (Thermo Fisher Scientific) offered signal space transformation-robust multi-array average for gene-level analysis using Thermo Fisher Scientific default analysis settings.
An equal amount (200 ng) of RNA prepared from the participants was mixed. Biotinylated cDNA was prepared from 100 ng of total RNA using the GeneChip WT PLUS Reagent Kit (Thermo Fisher Scientific, Waltham, MA) following the manufacturer’s instructions. Following fragmentation, 2 μg of single-stranded cDNA was hybridized for 16 h at 45°C on GeneChip Clariom S Array human (Thermo Fisher Scientific). Arrays were washed and stained in the GeneChip Fluidics Station 450 (Thermo Fisher Scientific). Clariom S array was scanned using the GeneChip Scanner 3000 7G (Thermo Fisher Scientific). Data were analyzed using the Expression Console Software 1.4 (Thermo Fisher Scientific) offered signal space transformation-robust multi-array average for gene-level analysis using Thermo Fisher Scientific default analysis settings.
8
Lipopolysaccharide-induced Inflammation Assay
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Crystallized Calloselasma rhodostoma venom, lipopolysaccharides from Escherichia coli O111:B4 (LPS), Histopaque 1077, 3,30-diaminobenzidine tablets, hydrogen peroxide 30%, ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), HEPES, Triton X-100, leupeptin, aprotinin, phenylmethylsulfonyl fluoride (PMSF), sodium orthovanadate, protease and phosphatase inhibitor cocktail, ethylenediaminetetraacetic acid disodium salt dihydrate (Na2 EDTA), bicinchoninic acid protein assay Kit (BCA), oil Red O, AACOCF3 (arachidonyl trifluoromethyl ketone), anti-mouse Fab specific-FITC and anti-β-actin were purchased from Sigma Chem. Co. (Misssouri, USA). CAY10650, A922500, prostaglandin E2 ELISA Kit, anti-COX-1 and anti-COX-2 were purchased from Cayman Chemical (Michigan, USA). Anti-cPLA2-α, anti-PTGES and anti-p-cPLA2-α were purchased from Santa Cruz Biotechnology (Texas, USA). PureLink kit, SuperScript III Reverse Transcriptase, GeneChip WT PLUS Reagent Kit and GeneChip Clariom S Array Human were purchased from Thermo Fisher Scientific (Massachusetts, USA). iTaq Universal SYBR Green Supermix were purchased from Bio-Rad (California, USA). All salts and reagents used obtained from Merck Millipore (Darmstadt, Germany) with low endotoxin or endotoxin-free grades.
9
Gene Expression Profiling of Human, Mouse, and Rat
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The RNA selected were amplified and hybridized using the “GeneChip® WT PLUS Reagent Kit” (Thermo Fisher Scientific, Santa Clara, CA, USA). Amplification was carried out from a total of 55 nanograms of initial RNA, and then the procedures described in the “GeneChip® WT PLUS Reagent Kit” were followed.
Amplification of the cDNA was quantified, fragmented, marked, and prepared for hybridization from the GeneChip® Clariom S Human Array (Thermo Fisher Scientific) for human, mouse, and rat, with more than 20,0000 genes annotated for expression level measurement using 5.5 μg of the product of simple chain cDNA and following the protocols of the GeneChip® Fluidics Station 450 (Thermo Fisher Scientific). Finally, the analysis performed was normalized using the Robust Multiarray Average (RMA) method.
Amplification of the cDNA was quantified, fragmented, marked, and prepared for hybridization from the GeneChip® Clariom S Human Array (Thermo Fisher Scientific) for human, mouse, and rat, with more than 20,0000 genes annotated for expression level measurement using 5.5 μg of the product of simple chain cDNA and following the protocols of the GeneChip® Fluidics Station 450 (Thermo Fisher Scientific). Finally, the analysis performed was normalized using the Robust Multiarray Average (RMA) method.
10
Transcriptome-wide Gene Expression Profiling
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Gene expression profiling was performed using Clariom™ S Pico human assay (Applied Biosystems™; ThermoFisher Scientific, Inc.; Foster City, CA, USA), which accurately measured the expression levels of more than 20,000 well-annotated genes, in order to obtain a transcriptome-wide gene-level expression profile. Sixteen samples were used in this exploratory analysis due to budget restrictions. Briefly, RNA was amplified and labeled using the GeneChip® WT PLUS Reagent Kit (Thermo Fisher Scientific, Inc.; Waltham, MA, USA). Amplification was performed with 100 ng of total RNA input, following the procedures described in the WT PLUS Reagent Kit user manual. The amplified cDNA was quantified, fragmented, and labeled to hybridize to GeneChip® Clariom S Human Array (Thermo Fisher Scientific, Inc.), using 5.5 μg of single-stranded cDNA product and following manufacturers’ protocols. Washing, staining (GeneChip® Fluidics Station 450, Thermo Fisher Scientific, Inc.), and scanning (GeneChip® Scanner 3000, Thermo Fisher Scientific, Inc.) were performed following the protocols outlined in the user manual for cartridge arrays.
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