Genechip system

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The GeneChip system is a microarray platform used for gene expression analysis and genotyping. It allows researchers to study the expression levels of thousands of genes simultaneously. The system includes instruments, reagents, and software to enable the analysis of DNA and RNA samples.

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GeneChips (22 citations) GeneChip Scanner 3000 system (9 citations) GeneChip Fluidics Station 450 systems (8 citations) GeneChip System 3000 (6 citations) GeneChip® Rat Gene 1.0 ST Array System (3 citations) GCOS Affymetrix GeneChip Operating System (2 citations) GeneChip operating system 1.4 (2 citations) Affymetric GeneChip® System 3000Dx v.2 platform (2 citations) GeneChip® Rat Gene 2.0 ST Array System (2 citations) U95 GeneChip system (2 citations)

40 protocols using genechip system

Microarray Analysis of Gene Expression

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Three biological replicates were performed for each condition, resulting in a total of 90 arrays in this study (Supplementary Figure S8). Total RNA was prepared and microarray analysis was performed using an Affymetrix GeneChip^® system as described elsewhere (29 (link),40 (link)). A high-density DNA microarray was utilized with the Affymetrix GeneChip system, and data extraction was performed based on the finite hybridization model (41 (link),42 (link)) as previously described (29 (link),40 (link)). The raw expression data sets were subjected to normalization (43 (link)), resulting in a common mean value (logarithmic) in all data sets. To avoid potential noise caused by the small values, normalized expression data with values less than −1.5 were removed, resulting in a total of 3398 genes for subsequent analysis. The averaged expression value for each gene over the three biological replicates was used for computational analyses. Both the normalized expression data sets and the raw CEL files were deposited in the NCBI Gene Expression Omnibus database under the GEO Series accession number (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE55719).

Murakami Y., Matsumoto Y., Tsuru S., Ying B.W, & Yomo T. (2015). Global coordination in adaptation to gene rewiring. Nucleic Acids Research, 43(2), 1304-1316.

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Gene Expression Analysis of Invasive Cancer Cells

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Total RNAs were extracted from the parental OC3 and highly invasive OC3IV cells for gene expression analysis using Affymetrix GeneChip system (Affymetrix, Santa Clara, CA, USA) and the microarray statistical analysis was performed by the Microarray Core Laboratory at the National Health and Research Institute (NHRI, Taiwan).

Jang T.H., Huang W.C., Tung S.L., Lin S.C., Chen P.M., Cho C.Y., Yang Y.Y., Yen T.C., Lo G.H., Chuang S.E, & Wang L.H. (2022). MicroRNA-485-5p targets keratin 17 to regulate oral cancer stemness and chemoresistance via the integrin/FAK/Src/ERK/β-catenin pathway. Journal of Biomedical Science, 29, 42.

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Microarray Gene Expression Analysis

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A microarray gene expression analysis was performed using a GeneChip® system with a Human Genome U133-plus 2.0 array, which was spotted with 54,675 probe sets (Affymetrix, Inc., Santa Clara, CA, USA) according to the manufacturer’s instructions. Total RNA from three experiments was pooled, and 500 ng of the RNA was used to synthesize cRNA with a GeneChip® 3′IVT Expression Kit (Affymetrix, Inc.). The array was hybridized with biotin-labeled cRNA at 45 °C for 16 h. After treatment with phycoerythrin-labeled streptavidin, the array was scanned using a probe array scanner (Affymetrix GeneChip® Scanner 3000). The obtained hybridization intensity data were further analyzed using GeneSpring^® GX (Agilent Technologies, Inc.) to extract the significant genes and Ingenuity^® Pathway Analysis tools (Tomy Digital Biology, Co., Ltd., Tokyo, Japan) to examine gene ontology, including biological processes, cellular components, molecular functions, and gene networks^{31 (link),32 (link)}. The microarray data were deposited in the Gene Expression Omnibus: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE136127.

Makino T., Mizawa M., Yoshihisa Y., Yamamoto S., Tabuchi Y., Miyai M., Hibino T., Sasahara M, & Shimizu T. (2020). Trichohyalin-like 1 protein plays a crucial role in proliferation and anti-apoptosis of normal human keratinocytes and squamous cell carcinoma cells. Cell Death Discovery, 6, 109.

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Transcriptome Analysis of Drug-Resistant Cancer Cells

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Total RNA was harvested from different groups of cells as described above. First-strand cDNA was transcribed with a primer containing a T7 oligo (dT) sequence. Next, multiple copies of biotin-modified aRNA were synthesized using the double-stranded cDNA as templates. The labeled aRNA was fragmented prior to hybridization with GeneChip^® 3ʹ expression arrays. In the Affymetrix Genechip system, hybridization is performed using the Human Genome U133 Plus 2.0 Array. Differences in the gene expression profiles of treated and control HL60/ADR cells were then analyzed. A difference in gene expression was defined as a -fold change ≥2. After removing negative-flagged probes and data normalization using R’s “limma” package, differentially expressed genes were selected based on the fold-changes for functional enrichment using R programming (www.cran.r-project.org) and the NextBio platform (www.nextbio.com). Gene set enrichment analysis was performed using an in-house web tool, in which the main data sources are provided by MSigDB (Broad Institute), Reactome, BioCarta, and the Gene Ontology Consortium, in addition to gene sets collected from literature mining.

Huang K., Sun Z., Ding B., Jiang X., Wang Z., Zhu Y, & Meng F. (2019). Suppressing Hedgehog signaling reverses drug resistance of refractory acute myeloid leukemia. OncoTargets and therapy, 12, 7477-7488.

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Transcriptional Profiling of Melanoma Cell Lines

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Total RNA samples of paired melanoma cell lines IGR39 and IGR37 were extracted and sent to the IRB Functional Genomic Core External Service (Barcelona). Once samples had been reversed-transcribed and labeled, they were hybridized onto Affymetrix GeneChip Human Genome U133 Plus 2.0 arrays. Hybridization, washing, staining and scanning were performed using Affymetrix GeneChip system instruments and protocols. For breast and colon cancer cell lines, expression data were obtained from the Gene Expression Omnibus (GSE11683, GSE10843 and GSE57083). For qRT–PCR experiments, total RNA was extracted using Trizol reagent and retrotranscribed using the ThermoScript RT-PCR system (Invitrogen). The reaction was carried out following the methods for use of SYBR Green (Applied Biosystems), and B2M, GAPDH and ACTB were used as housekeeping genes to enable normalization. For primer sequences please refer to Supplementary Table 3. We performed reactivation treatments with the demethylating agent 5-aza-2′-deoxycytidine (AZA; Sigma) at 0.5 μM for 72 h. For immunoblotting assays, we extracted total protein using RIPA (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA and EGTA, 1% NP40, 0.5% of sodium deoxycholate, 0.1% of SDS, and protease and phosphatase inhibitors from Roche). All the antibodies used in this study are described in Supplementary Table 3.

Vizoso M., Ferreira H.J., Lopez-Serra P., Javier Carmona F., Martínez-Cardús A., Girotti M.R., Villanueva A., Guil S., Moutinho C., Liz J., Portela A., Heyn H., Moran S., Vidal A., Martinez-Iniesta M., Manzano J.L., Fernandez-Figueras M.T., Elez E., Muñoz-Couselo E., Botella-Estrada R., Berrocal A., Pontén F., van den Oord J., Gallagher W.M., Frederick D.T., Flaherty K.T., McDermott U., Lorigan P., Marais R, & Esteller M. (2015). Epigenetic activation of a cryptic TBC1D16 transcript enhances melanoma progression by targeting EGFR. Nature medicine, 21(7), 741-750.

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Total RNA Extraction and Gene Expression Analysis

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The total RNA was extracted from each of the five above-mentioned cell groups as described in a previous report^{19 (link)}. The total RNAs were extracted using the RNeasy^® Micro Kit (Qiagen, Bothell, Washington, USA); their concentration and purity were determined using a bioanalyzer (Agilent Technologies, Santa Clara, California, USA) according to the manufacturer's instructions. The gene expression was analyzed using a GeneChip^® system with a Human Genome U133-plus 2.0 array (Affymetrix, Santa Clara, California, USA) according to the manufacturer's instructions. The scanned chip was analyzed using the GeneChip^® Analysis Suite software program Affymetrix) and obtained hybridization intensity data.

Sato Y., Yoshino H., Ishikawa J., Monzen S., Yamaguchi M, & Kashiwakura I. (2023). Prediction of hub genes and key pathways associated with the radiation response of human hematopoietic stem/progenitor cells using integrated bioinformatics methods. Scientific Reports, 13, 10762.

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Identification of miRNA Target Genes

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The RNA samples subjected to miRNA microarray analysis were also used to identify putative miRNA target genes. Briefly, total RNA from the 3 undifferentiated and 3 decidualized cultures were combined separately; and the two sets of mRNAs subjected to gene expression microarrays. Total RNA was reverse-transcribed, amplified and labelled using a GeneChip^® 3′IVT Expression Kit (Affymetrix Inc., Santa Clara, CA, USA). The complementary RNA (cRNA) was purified, fragmented according to the manufacturer’s protocol and hybridised on the Affymetrix GeneChip^® Human Genome U133 Plus 2.0 array. Hybridisation, washing, staining and scanning were performed using the Affymetrix GeneChip^® system instruments and protocols. The expression levels of each gene obtained from the scanned data were normalised among samples using the robust multi-array average (RMA) method. The mRNAs that were differentially upregulated (>2-fold) or downregulated (<0.5-fold) upon decidualization were extracted.

Tochigi H., Kajihara T., Mizuno Y., Mizuno Y., Tamaru S., Kamei Y., Okazaki Y., Brosens J.J, & Ishihara O. (2017). Loss of miR-542-3p enhances IGFBP-1 expression in decidualizing human endometrial stromal cells. Scientific Reports, 7, 40001.

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Microarray Analysis of Mouse Transcriptome

Cited 2 times

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Microarray analysis was performed using a GeneChip^® system with a Mouse Genome 430 2.0 array (Affymetrix, Inc.) spotted with 45,101 probe sets. Array samples were prepared as described in the Affymetrix GeneChip^® Expression Technical Manual. Total RNA from three experiments was pooled, and 500 ng of the RNA was used to synthesize cRNA with a GeneChip^® 3′ IVT Express Kit (Affymetrix, Inc.). The array was hybridized with biotin-labeled cRNA at 45°C for 16 h. After the treatment with phycoerythrin-labeled streptavidin, the array was scanned using a prove array scanner (Affymetrix GeneChip^® Scanner 3000). The obtained hybridization intensity data were further analyzed using GeneSpring^® GX (Agilent Technologies, Inc.) to extract the significant genes and Ingenuity^® Pathway Analysis tools (Tomy Digital Biology, Co., Ltd.) to examine gene ontology, including biological processes, cellular components, molecular functions, and gene networks (21 (link),27 (link)).

Tabuchi Y., Hasegawa H., Suzuki N., Furusawa Y., Hirano T., Nagaoka R., Hirayama J., Hoshi N, & Mochizuki T. (2020). Genetic response to low-intensity ultrasound on mouse ST2 bone marrow stromal cells. Molecular Medicine Reports, 23(3), 173.

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Microarray Analysis of miRNA-6852 in HEK293

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DNA microarray assay was performed using the Affymetrix GeneChip System (Affymetrix). The Affymetrix Human Exon 1.0 ST Array containing 1.4 million probe sets was used. Total cellular RNA was extracted from HEK293 cells transfected with Negative miRNA mimic and mimic miRNA-6852 by using phenol-chloroform based extraction using Qiazol (Qiagen) and quantitated following the manufacturer’s protocols (Affymetrix). Terminal labeling and hybridization, array wash, stain, and scan were processed according to the Affymetrix recommended standard protocol. Intensity data were processed and summarized to gene level with Partek (Partek). Differentially expressed gene candidates were selected for verification with an absolute fold change difference >2.0.

Poudyal D., Herman A., Adelsberger J.W., Yang J., Hu X., Chen Q., Bosche M., Sherman B.T, & Imamichi T. (2018). A novel microRNA, hsa-miR-6852 differentially regulated by Interleukin-27 induces necrosis in cervical cancer cells by downregulating the FoxM1 expression. Scientific Reports, 8, 900.

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Transcriptome Analysis Using Affymetrix GeneChip

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Transcriptome analysis was performed using the Affymetrix GeneChip system according to the manufacturer’s instructions as described^{46 (link)}. Labeled cRNA probes were hybridized to Mouse Genome 430 2.0 array (Affymetrix) and scanned by GeneChIP scanner 3000 (Affymetrix). To calculate the average difference for each gene probe, GeneChip Analysis Suite software version 5.0 (Affymetrix) was used.

Matsumura Y., Ito R., Yajima A., Yamaguchi R., Tanaka T., Kawamura T., Magoori K., Abe Y., Uchida A., Yoneshiro T., Hirakawa H., Zhang J., Arai M., Yang C., Yang G., Takahashi H., Fujihashi H., Nakaki R., Yamamoto S., Ota S., Tsutsumi S., Inoue S.I., Kimura H., Wada Y., Kodama T., Inagaki T., Osborne T.F., Aburatani H., Node K, & Sakai J. (2021). Spatiotemporal dynamics of SETD5-containing NCoR–HDAC3 complex determines enhancer activation for adipogenesis. Nature Communications, 12, 7045.

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