Paired differential expression analysis between brown and white samples assayed with GeneChip PrimeView (Affymetrix, Santa Clara, CA) arrays was performed in limma (Ritchie et al., 2015 ).
Genechip primeview
Manufactured by Thermo Fisher Scientific
Sourced in United States
The GeneChip PrimeView is a high-density oligonucleotide array that provides comprehensive coverage of the human transcriptome. It is designed to analyze the expression of protein-coding and non-coding transcripts. The core function of the GeneChip PrimeView is to enable gene expression profiling and analysis.
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Lab products found in correlation
Genechip scanner 3000 7g, by Thermo Fisher Scientific (3 mentions)
Rneasy kit, by Merck Group (2 mentions)
Ingenuity pathway analysis ipa, by Qiagen (2 mentions)
Agilent rna 6000 nano kit, by Agilent Technologies (2 mentions)
Genechip 3 ivt expression kit, by Thermo Fisher Scientific (1 mentions)
Expression console, by Thermo Fisher Scientific (1 mentions)
Affymetrix scanner 3000, by Thermo Fisher Scientific (1 mentions)
Agilent 2100, by Agilent Technologies (1 mentions)
Genechip fluidics station 450, by Thermo Fisher Scientific (1 mentions)
Affymetrix genechip scanner 3000, by Thermo Fisher Scientific (1 mentions)
Genespring 11, by Agilent Technologies (1 mentions)
Enzo kit, by Thermo Fisher Scientific (1 mentions)
Feature extraction software 10, by Agilent Technologies (1 mentions)
Experion, by Bio-Rad (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Nanodrop 2000, by Agilent Technologies (1 mentions)
Genechip command console software, by Thermo Fisher Scientific (1 mentions)
Mouse gene 2.0 st genechip, by Thermo Fisher Scientific (1 mentions)
Rneasy spin column kit, by Qiagen (1 mentions)
10 protocols using genechip primeview
1
Differential Expression Analysis of Brown and White Samples
2
Transcriptomic Analysis of Tumor Xenografts
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Total RNA was amplified and fragmented using a GeneChip® 3′ IVT expression kit (Affymetrix, Santa Clara, CA). Then the samples were hybridized onto a GeneChip® PrimeView™ human gene expression array (Affymetrix). Arrays were scanned on an Affymetrix GeneChip® scanner 3000 7G (Affymetrix). Resulting data was subject to bioinformatics analysis. Briefly, the raw CEL data were processed on an Expression Console™ (version 1.1, Affymetrix). Signal intensities were normalized by the robust multiarray average normalization approach. On 9 pairs of samples which consisted of original patient samples and their corresponding xenograft tumors, unsupervised hierarchical clustering analysis was performed by ‘hclust’ package on R with criteria Euclidian distance and average linkage.
3
Microarray Analysis and Gene Expression
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Microarray analysis was accomplished by hybridization to a GeneChip PrimeView human gene expression array per manufacturer's instructions (Affymetrix, Santa Clara, CA). Genes that underwent at least a 2-fold change between treatment groups were selected for real-time PCR validation.
4
RNA Sequencing of Gene Expression in Transfected SGC-7901 Cells
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Gene expression in transfected SGC-7901 cells was detection by RNA screening analysis in Shanghai Bioscienceres, Co., Ltd. (Shanghai, China). Briefly, total RNA was extracted by the RNeasy kit (Sigma, St. Louis, MO, USA). Quality and integrity of total RNA was determined by Nanodrop 2000 (Thremo Fisher Scientific, Waltham, MA, USA) and Agilent 2100 and Agilent RNA 6000 Nano Kit (Agilent, Santa Clara, CA, USA). RNA sequencing was performed with Affymetrix human GeneChip PrimeView according to the manufacturer’s instruction and the outcomes were scanned by Affymetrix Scanner 3000 (Affymetrix, Santa Clara, CA, USA). Raw data statistical significance assessment was accomplished using a Welch t-test with Benjamini-Hochberg FDR (|Fold Change| ≥ 2.0 and FDR < 0.05 as significant). Significant difference analysis and functional analysis based on Ingenuity Pathway Analysis (IPA) (Qiagen, Hilden, Germany) was executed, and |Z - score| > 2 is considered meaningful.
5
Transcriptional Profiling Using GeneChip Array
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To investigate the transcriptional profiles of the samples, we used a GeneChip PrimeView and a Human Gene Expression Array GeneChip 3 IVT PLUS Reagent Kit (Affymetrix, USA). Total RNA (100 ng) was used for a reverse transcription reaction in the presence of a poly(A) binding proteins to generate gene expression profiles from mRNA. RNA amplification is based upon linear amplification and employs T7 in vitro transcription (IVT) technology. In the second step, single-stranded cDNA is converted to double-stranded cDNA, which acts as a template for in vitro transcription. The reaction uses DNA polymerase and RNase H to simultaneously degrade the RNA and synthesize second-strand cDNA. Labeled complementary RNA (cRNA) was synthesized and amplified via IVT of the second-stranded cDNA template using T7 RNA polymerase, followed by purification, fragmentation, and hybridization to the GeneChip PrimeView array. Hybridization was performed in the GeneChip Hybridization Oven 645 instrument at a temperature to 45°C with rotation at 60 RPM for 16 hours, followed by washing in the GeneChip Fluidics Station 450 and scanning using a laser scanner (GeneChip Scanner 3000 7G, Affymetrix). Finally, the data were collected using Expression Console software.
6
Transcriptome Profiling via Affymetrix Microarray
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DNA microarray analysis was performed as described previously [37] (link) using GeneChip® PrimeView™ human gene expression array (Affymetrix, Santa Clara, CA) following the standard Affymetrix protocol.
7
Genome-wide Gene Expression Profiling of OVAT Cells
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The whole Human Gene Expression Array (Affymetrix GeneChip® PrimeView™, Homo sapiens; Affymetrix, Santa Clara, CA, USA) was used to screen for gene expression in the OVAT cells of pregnant women according to the manufacturer's instructions.
First, the ribosomal RNA was removed from the total RNA, and the purified mRNA was amplified and reversely transcribed into fluorescent/biotinylated complementary (c)DNA using the ENZO kit (Affymetrix). The cDNA was fragmented and then hybridized to the Affymetrix GeneChip® PrimeView™ containing 36,000 probe-sets representing ~20,000 unique genes. Six microarrays were run for the samples (from the six patients including three cases with normal glucose tolerance and three cases with GDM mentioned above) tested and each array was replicated twice. Subsequently, the arrays were processed on an Affymetrix fluidics station (Affymetrix), where they were subjected to automated washing and staining. The arrays were scanned using an Affymetrix GeneChip scanner 3000 (Affymetrix), raw data were obtained using the Feature Extraction Software 10.7 (Agilent Technologies, Inc.) and normalized using the quantile algorithm with Gene Spring 11.0 (Agilent Technologies, Inc.) to remove background bias with the normalization value set to 1. Systematic bioinformatic analyses of the microarray data were performed by Novel Bioinformatics Co., Ltd. (Shanghai, China).
First, the ribosomal RNA was removed from the total RNA, and the purified mRNA was amplified and reversely transcribed into fluorescent/biotinylated complementary (c)DNA using the ENZO kit (Affymetrix). The cDNA was fragmented and then hybridized to the Affymetrix GeneChip® PrimeView™ containing 36,000 probe-sets representing ~20,000 unique genes. Six microarrays were run for the samples (from the six patients including three cases with normal glucose tolerance and three cases with GDM mentioned above) tested and each array was replicated twice. Subsequently, the arrays were processed on an Affymetrix fluidics station (Affymetrix), where they were subjected to automated washing and staining. The arrays were scanned using an Affymetrix GeneChip scanner 3000 (Affymetrix), raw data were obtained using the Feature Extraction Software 10.7 (Agilent Technologies, Inc.) and normalized using the quantile algorithm with Gene Spring 11.0 (Agilent Technologies, Inc.) to remove background bias with the normalization value set to 1. Systematic bioinformatic analyses of the microarray data were performed by Novel Bioinformatics Co., Ltd. (Shanghai, China).
8
Abdominal Tissue RNA Profiling
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RNA from each abdominal biopsy sample was isolated using the Qiagen RNeasy Mini Kit (Cat No. 74104). The quantification of samples was done using a Microfluidic-based capillary electrophoresis system (Bio-Rad Experion, Bio-Rad Laboratories, Inc., Philadelphia, PA, USA). Total RNA was made to undergo reverse transcription to synthesize the first-strand cDNA. The cDNA, thus formed was then converted to a double-stranded cDNA template during second-strand cDNA synthesis. Biotinylated ribonucleotide was subsequently incorporated by in vitro transcription reaction and then purified by the bead-based purification method. The purified biotin-labeled-cRNA was then fragmented using a fragmentation buffer, and then the sample was hybridized onto Affymetrix GeneChipPrimeView (Affymetrix Inc., Santa Clara, CA, USA). The obtained CEL files per sample were subjected to normalization and non-specific filtering using Bioconductor packages affy and genefilter. As primeview annotation package primeview.db was not available at Bioconductor, annotations file “PrimeView.na36.annot” was downloaded from the Affymetrix website, and the package was created using AnnotationForge and Human.db0 packages. Raw CEL files and series matrix files have been submitted to NCBI Genomic Expression Omnibus database (GEO Accession # GSE78721).
9
Transcriptomic Profile of Eca-109 Cells with MEX3A Knockdown
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The detection of gene expression profile based on Eca-109 cells with shMEX3A or shCtrl by RNA screening analysis was completed by Shanghai Bioscienceres, Co., Ltd. (Shanghai, China). Briefly, total RNA from Eca-109 cells was extracted by the RNeasy kit (Sigma, St. Louis, MO, USA). Total RNA quality was evaluated by Agilent RNA 6000 Nano Kit on Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) and quantified using Thremo Nanodrop 2000 (Waltham, MA, USA). RNA sequencing was performed human GeneChip primeview (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s instruction and the outcomes were scanned by GeneChip Scanner Scanner 3000 (Affymetrix, Santa Clara, CA, USA). Raw data statistical significance assessment was accomplished using a Welch t-test with Benjamini-Hochberg FDR (FDR < 0.05 as significant). Ingenuity Pathway Analysis (IPA) (Qiagen, Hilden, Germany) was executed for all significant differentially expressed genes. |Z - score| > 2 is considered meaningful.
10
Transcriptome Analysis of RNA Expression
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Total RNA was isolated using the RNeasy spin column kit (Qiagen) and quantified using a BioSpectrometer (Eppendorf). One hundred nanograms of RNA was used as input for the GeneChip® PrimeView™ (3′ IVT human array) and GeneChip™ Mouse Gene 2.0 ST (mouse exon array) microarrays (Affymetrix). Synthesis, labeling, and purification of biotinylated complementary RNA and sense-stranded DNA targets were carried out according to manufacturers’ instructions using the GeneChip™ 3′ IVT PLUS Reagent Kit and GeneChip™ WT PLUS Reagent Kit, respectively. Hybridization was performed using a GeneChip Hybridization Oven 640 overnight at 45 °C. Microarray washing and staining was performed on a GeneChip Fluidics Station 450 and scanning on a GeneChip Scanner 3000 7G, commanded by the Affymetrix GeneChip Command Console software. Probe-level analysis including background subtraction and quantile normalization took place with the Robust Multi Array Average Algorithm using the Affymetrix Expression Console Software 1.3. DEGs (p < 0.05 and fold change > 1.5 for the GeneChip™ Mouse Gene 2.0 ST exon array and p < 0.01 and fold change > 1.5 for the GeneChip® PrimeView™) were determined using the Transcriptome Analysis Console v3.0. Raw and processed Affymetrix data have been deposited in the Gene Expression Omnibus repository under accession number GSE120127.
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