Sureprint g3 human gene expression 8 60 k v2 microarray

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The SurePrint G3 Human Gene Expression 8 × 60 K v2 Microarray is a high-density microarray designed for comprehensive whole-genome expression profiling of human genes. It provides a comprehensive coverage of the human transcriptome with over 60,000 probes.

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Gene expression microarrays (4 citations) Agilent G4851B SurePrint G3 Human Gene Expression 8 × 60 K v2 microarrays (2 citations)

28 protocols using sureprint g3 human gene expression 8 60 k v2 microarray

Gene Expression Profiling of Cell Lines

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Gene expression profiles of the wild-type and mutant cell lines were measured using SurePrint G3 Human Gene Expression 8 × 60 Kv2 Microarray (Agilent Technologies, Santa Clara, CA, USA) following the manufacturer’s protocol and are described in detail elsewhere ([49 (link)] and references therein). Background-corrected expression values were extracted using Agilent Feature Extraction Software. Data was log-transformed and quantile normalized using Agilent GeneSpring Software. Fold-changes for all genes were calculated as ratio of mutant vs. wild-type.

Menegatti J., Nakel J., Stepanov Y.K., Caban K.M., Ludwig N., Nord R., Pfitzner T., Yazdani M., Vilimova M., Kehl T., Lenhof H.P., Philipp S.E., Meese E., Fröhlich T., Grässer F.A, & Hart M. (2022). Changes of Protein Expression after CRISPR/Cas9 Knockout of miRNA-142 in Cell Lines Derived from Diffuse Large B-Cell Lymphoma. Cancers, 14(20), 5031.

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Gene Expression Profiling of Tumor Samples

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Purified total RNA for GEP was then amplified and fluorescently labeled using a One-Color Low Input Quick Amp Labeling Kit (Agilent Technologies). Cy3-labeled cRNAs were hybridized to a SurePrint G3 Human Gene Expression 8 × 60K v2 Microarray (Agilent Technologies). A signature analysis based on the gene expression was performed using the expression ratio of the tumor and the corresponding normal tissue (T/N). The expression signature/score was calculated from the average of genes in unique gene sets corresponding to each individual signature (TP53 inactivation^{28 (link)}, chromosomal instability [CIN]^{49 (link)}).

Imamura T., Ashida R., Ohshima K., Uesaka K., Sugiura T., Ohgi K., Yamada M., Otsuka S., Hatakeyama K., Nagashima T., Sugino T., Urakami K., Akiyama Y, & Yamaguchi K. (2023). Characterization of pancreatic cancer with ultra-low tumor mutational burden. Scientific Reports, 13, 4359.

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Differential Expression Profiling of CRC

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Three pairs of CRC tumor tissues and MLNs were used to synthesize double-stranded complementary DNA (cDNA), which was labeled and hybridized on the SurePrint G3 Human Gene Expression 8×60K v2 Microarray (Agilent Technologies, Santa Clara, CA, USA). Processed slides were scanned with the Agilent G2505C Microarray Scanner (Agilent Technologies) after hybridization and washing. Raw data were extracted using Feature Extraction (version 10.7.1.1; Agilent Technologies). Then, quantification of normalization and subsequent data processing were performed using the GeneSpring software (version 12.0; Agilent Technologies). After that, raw signals from the microarray were log₂ transformed and specific expression of mRNAs and lncRNAs were defined when the absolute value of fold change was >2 and P-value was <0.05. The microarray profiling was conducted by the OE Biotechnology Company (Shanghai, People’s Republic of China).

Yang P., Xu Z.P., Chen T, & He Z.Y. (2016). Long noncoding RNA expression profile analysis of colorectal cancer and metastatic lymph node based on microarray data. OncoTargets and therapy, 9, 2465-2478.

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Gene Expression Profiling of Tumor Samples

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GEP analysis was performed in 892 (98.0%) samples, as previously described.14, 17 Total RNA was extracted from approximately 10‐mg tissue using an miRNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. RNA samples with an RNA integrity number of greater than or equal to 6 were used for DNA microarray analysis. Briefly, total RNA (100 ng) was amplified and fluorescently labeled. Labeled samples were hybridized to a SurePrint G3 Human Gene Expression 8 × 60 K v2 Microarray (Agilent Technologies, Santa Clara, CA). Data analysis was performed using GeneSpring GX software (Agilent Technologies). Raw signal intensity values were log transformed and normalized to the 75th percentile. The fold change between tumor and normal tissues from the same patient was calculated from the normalized values.

Hino H., Shiomi A., Kusuhara M., Kagawa H., Yamakawa Y., Hatakeyama K., Kawabata T., Oishi T., Urakami K., Nagashima T., Kinugasa Y, & Yamaguchi K. (2019). Clinicopathological and mutational analyses of colorectal cancer with mutations in the POLE gene. Cancer Medicine, 8(10), 4587-4597.

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RNA Extraction and Microarray Analysis of IAV-Infected Calu-3 Cells

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RNA extraction from IAV- and mock-infected Calu-3 cells in quadruplicate was performed as previously described (41 (link)). Probe labeling and microarray slide hybridization for each biological replicate were performed using a SurePrint G3 Human Gene Expression 8×60K v2 microarray (Agilent Technologies) according to the manufacturer’s instructions. Slides were scanned on an Agilent DNA microarray scanner (model G2505B) using the XDR setting, and raw images were analyzed using the Agilent Feature Extraction software program (version 9.5.3.1). Extracted raw data were background corrected using the norm-exp method with an offset of 1 and quantile normalized using the limma software package (42 ) in the R environment. Replicated probes were mean summarized. All probes were required to pass Agilent quality control (QC) flags (“gIsFound,” “gIsWellAboveBG,” “gIsSaturated,” “gIsFeatNonUnifOL,” and “gIsFeatPopnOL”) for all replicates of at least one infected time point (29,382 probes passed QC filtering). For each sample, a log₂FC value was calculated as the difference between log₂-normalized data for this sample and the average of log₂-normalized data for time-matched mocks. Microarray annotation was retrieved from the GEO data bank (GEO accession no. GPL17077).

Josset L., Zeng H., Kelly S.M., Tumpey T.M, & Katze M.G. (2014). Transcriptomic Characterization of the Novel Avian-Origin Influenza A (H7N9) Virus: Specific Host Response and Responses Intermediate between Avian (H5N1 and H7N7) and Human (H3N2) Viruses and Implications for Treatment Options. mBio, 5(1), e01102-13.

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Bortezomib-induced gene expression changes

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Cells were treated with 10nM bortezomib for 6 and 24 hours. Total RNA isolation was performed using illustra RNAspin mini RNA isolation kit (GE Healthcare, Uppsala, Sweden) according to manufacturer's protocol. Gene expression microarrays were performed using Agilent Sure Print G3 Human gene expression 8×60K v2 microarray. The labeling was done according to manufacturer's protocol. Two biological replicates were used for microarray analysis. In the LP-1-cIAP2-eGFP cells, a lower expression of cIAP2 (BIRC3) was found compared to the control in the array. This is due to the design of the probe on the array to the 3 prime UTR detecting only the endogenous cIAP2. RT-qPCR analysis was used to verify that the LP-cIAP2-eGFP cells overexpress the cIAP2 mRNA as compared to control (Supplementary Figure 1).http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=kvqjyigmpxcpvid&acc=GSE63520

Duvefelt C.F., Lub S., Agarwal P., Arngården L., Hammarberg A., Maes K., Van Valckenborgh E., Vanderkerken K, & Wiklund H.J. (2015). Increased resistance to proteasome inhibitors in multiple myeloma mediated by cIAP2 - implications for a combinatorial treatment. Oncotarget, 6(24), 20621-20635.

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Gene Expression Profiling via Microarray

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Total RNA prepared using an RNeasy Mini Kit (Qiagen, Hilden, Germany) was reverse-transcribed, and the resultant cDNA was hybridized with SurePrint G3 Human Gene Expression 8×60K v2 microarray (Agilent Technologies, Santa Clara, CA, USA) at 65 °C for 17 h with gentle rotation. Gene expression profiling was performed by Medical & Biological Laboratories (Nagoya, Japan).

Makise M., Uchimura R., Higashi K., Mashiki Y., Shiraishi R., Shutoku Y, & Kuniyasu A. (2021). Overexpression of the nucleoporin Nup88 stimulates migration and invasion of HeLa cells. Histochemistry and Cell Biology, 156(5), 409-421.

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Human Gene Expression Microarray Analysis

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Total RNA was isolated and subjected to microarray analysis as described previously18 using SurePrint G3 Human Gene Expression 8 × 60K v2 Microarray (Agilent Technologies). RNA samples with RNA integrity number ≥5.9 were used for microarray analysis. Microarray analysis was carried out in accordance with the MIAME guidelines.27 Data analysis was carried out using GeneSpring GX (Agilent Technologies), Subio platform (Subio), and Microsoft Excel. Probes to be analyzed were selected according to the reference genome sequence, hg19, obtained from the UCSC Genome Browser.28 Raw signal intensity values were log‐transformed and normalized to the 75th percentile. Microarray data for mRNA expression are available through the NCBI database under accession http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE136755.

Ohshima K., Fujiya K., Nagashima T., Ohnami S., Hatakeyama K., Urakami K., Naruoka A., Watanabe Y., Moromizato S., Shimoda Y., Ohnami S., Serizawa M., Akiyama Y., Kusuhara M., Mochizuki T., Sugino T., Shiomi A., Tsubosa Y., Uesaka K., Terashima M, & Yamaguchi K. (2019). Driver gene alterations and activated signaling pathways toward malignant progression of gastrointestinal stromal tumors. Cancer Science, 110(12), 3821-3833.

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Detailed Protocol for Quantitative RT-PCR and Microarray Analysis

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Detailed methods for real-time quantitative RT-PCR (qRT-PCR) and information about primers and probes (Supplementary Table E4) are shown in this article’s Online Repository. The mRNA expression levels were normalized to a housekeeping gene, β-glucuronidase (GUSB).
Microarray analysis with SurePrint G3 Human Gene Expression 8×60K v2 Microarray (Agilent Technologies) was performed according to the manufacturer’s instructions at University of North Carolina Lineberger Comprehensive Cancer Center, within the Genomics Core Facility. The data were log 2 transformed, normalized by global normalization, centered and then analyzed using Subio Platform ver. 1.23 (Subio Inc., Kagoshima, Japan). Gene ontology (GO) enrichment analysis and pathway analysis were performed by g:Profiler. GO and pathways associated with endotypes were identified based on an adjusted p value <0.05 by g:SCS algorithm.^{17 (link)}

Klingler A.I., Stevens W.W., Tan B.K., Peters A.T., Poposki J.A., Grammer L.C., Welch K.C., Smith S.S., Conley D.B., Kern R.C., Schleimer R.P, & Kato A. (2020). Mechanisms and biomarkers of inflammatory endotypes in chronic rhinosinusitis without nasal polyps. The Journal of allergy and clinical immunology, 147(4), 1306-1317.

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Gene Expression Profiling using Microarray

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Purified total RNA for gene expression profiling was amplified and fluorescently labeled using a One‐Color Low Input Quick Amp Labeling Kit (Agilent Technologies) according to the manufacturer's instructions. Hybridization and scanning were carried out as in previous reports.28, 29 Cy3‐labeled cRNAs were hybridized to a SurePrint G3 Human Gene Expression 8 × 60K v2 Microarray (Agilent Technologies), which contained 50 599 probes representing 29 833 genes registered in the Entrez Gene Database (https://www.ncbi.nlm.nih.gov/gene). After hybridization and washing, the fluorescence was scanned using a DNA Microarray Scanner (Agilent Technologies), and then assessed by Agilent Feature Extraction software.

Hatakeyama K., Nagashima T., Ohshima K., Ohnami S., Ohnami S., Shimoda Y., Serizawa M., Maruyama K., Naruoka A., Akiyama Y., Urakami K., Kusuhara M., Mochizuki T, & Yamaguchi K. (2019). Mutational burden and signatures in 4000 Japanese cancers provide insights into tumorigenesis and response to therapy. Cancer Science, 110(8), 2620-2628.

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