Agilent s dna microarray scanner

Manufactured by Agilent Technologies

Sourced in United States

Agilent's DNA microarray scanner is a high-performance instrument designed for the analysis of DNA microarray data. It captures and digitizes fluorescent signals from labeled DNA samples hybridized to microarray surfaces.

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6 protocols using agilent s dna microarray scanner

Bovine Microarray Hybridization Protocol

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For microarrays, the synthesis of target cRNA probes and hybridization were performed using Agilent’s Low RNA Input Linear Amplification kit (Agilent Technologies, Palo Alto, CA, USA) according to the manufacturer’s instructions. The fragmented cRNA was resuspended with 2X hybridization buffer and directly pipetted onto the assembled Agilent’s Bovine Oligo Microarray (44K). The arrays were hybridized at 65°C for 17 h using the Agilent Hybridization oven and the hybridized microarrays were washed as described in the manufacturer’s washing protocol (Agilent Technologies).
The hybridized images were scanned using Agilent’s DNA microarray scanner and quantified with Feature Extraction Software (Agilent Technologies). All data normalization and selection of fold-changed genes were performed using GeneSpringGX 7.3 (Agilent Technologies). The averages of normalized ratios were calculated by dividing the average of the normalized signal channel intensity by the average of the normalized control channel intensity. Hierarchical clustering was performed with TIGR MeV Ver.4.9 software (Institute of Genomic Research, Rockville, MD, USA) [26 (link)]. Microarray data are available from Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) with the accession number GSE92672.

Kim D., Jung Y.G, & Roh S. (2017). Microarray analysis of embryo-derived bovine pluripotent cells: The vulnerable state of bovine embryonic stem cells. PLoS ONE, 12(3), e0173278.

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Pancreatic Islet Isolation and Analysis

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Islets were isolated from mice as previously described (Liadis et al, 2005 (link); Choi et al, 2010 (link)), using 3 ml of 3 mg/ml collagenase (Sigma‐Aldrich) injected into the common bile duct for pancreatic digestion, and islets were picked up under dissecting microscope in RPMI 1640 supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin, and frozen immediately. RNA was extracted using NucleoSpin^® RNA II, Macherey‐Nagel and underwent PC and microarray analysis by Miltenyi Biotec. Briefly, 15 ng of each total RNA sample was used to produce Cy3(control samples)‐ and Cy5(experimental samples)‐labeled cRNA, the RNA samples were amplified and labeled using Agilent Low Input Quick Amp Labeling Kit (Agilent Technologies). Next, 300 ng of the corresponding Cy3‐ and Cy5‐labeled fragmented cRNA were combined and hybridized overnight (65°C) to Agilent Whole Mouse Genome Oligo Microarrays 8 × 60 K. Fluorescence signals of the hybridized Agilent Oligo Microarrays were detected using Agilent’s DNA microarray scanner (Agilent Technologies).

Jiang Z., Li H., Schroer S.A., Voisin V., Ju Y., Pacal M., Erdmann N., Shi W., Chung P.E., Deng T., Chen N., Ciavarra G., Datti A., Mak T.W., Harrington L., Dick F.A., Bader G.D., Bremner R., Woo M, & Zacksenhaus E. (2022). Hypophosphorylated pRb knock‐in mice exhibit hallmarks of aging and vitamin C‐preventable diabetes. The EMBO Journal, 41(4), e106825.

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Comprehensive aCGH for Hematological Cancer

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DNA was extracted from fresh biopsy material or cytogenetic fixed cell suspension by QIAmp DNA Blood Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s recommendation. For aCGH analysis CytoSureTM Haematological Cancer and SNP Array (8x60k) (Oxford Gene Technology (OGT), Yarnton, Oxford OX5 1PF UK) was used. On this array average gene resolution was 68 Kb and SNP resolution was equal 30 Mb. The procedure of aCGH was performed following the manufacturer’s protocol. The reference DNA was from two pools of normal individuals (male and female), run as a same-sex control. Each patient and reference DNA was labeled with Cy3 and Cy5, respectively. Purification of labeled products, hybridization, and post-wash of the array was carried out according to OGT’s recommendation and with their proprietary solutions. Array slides were scanned with Agilent’s DNA Microarray Scanner and extraction software (Agilent, Santa Clara, USA).

Grygalewicz B., Woroniecka R., Rygier J., Borkowska K., Rzepecka I., Łukasik M., Budziłowska A., Rymkiewicz G., Błachnio K., Nowakowska B., Bartnik M., Gos M, & Pieńkowska-Grela B. (2016). Monoallelic and biallelic deletions of 13q14 in a group of CLL/SLL patients investigated by CGH Haematological Cancer and SNP array (8x60K). Molecular Cytogenetics, 9, 1.

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Transcriptomic Analysis by Microarray

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For transcriptomic analysis, microarray was performed as follows as recommended by E-Biogen (Seoul, Korea): Total RNA quality was controlled using an Agilent 2100 Bioanalyzer instrument (Agilent, CA, USA). Amplification and labeling of RNA was carried out with Agilent’s Low RNA Input linear amplification kit PLUS (Agilent). Microarray hybridization and sample washing were conducted using Agilent’s Gene Expression Hybridization Kit (Agilent) and Agilent’s Gene Expression Wash Buffer Kit (Agilent), respectively. Following this pre-process, samples were analyzed with Agilent’s DNA microarray scanner and Feature Extraction software (Agilent).

Na J., Choi S.A., Khan A., Huh J.Y., Piao L., Hwang I., Ha H, & Park Y.H. (2019). Integrative Omics Reveals Metabolic and Transcriptomic Alteration of Nonalcoholic Fatty Liver Disease in Catalase Knockout Mice. Biomolecules & Therapeutics, 27(2), 134-144.

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miRNA Profiling of HT29 Cells Treated with Ptero and/or MLT

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Based on the paper of [13 (link)], for control and test RNAs isolated from HT29 cells exposed to Ptero and/or MLT for 24 h, the syntheses of target miRNA probes and hybridization were conducted by using Agilent’s miRNA Labeling Reagent and Hybridization kit. Total RNAs (100 ng each) were dephosphorylated, denatured, and incubated for 10 min at 100 °C, ligated with pCp-Cy3 mononucleotide and purified with MicroBioSpin 6 columns (Bio-rad, Hercules, CA, USA). After purification, denatured labelled probes were transferred onto assembled Agilent Human miRNA Microarray (Human miRNA Microarray Release, AXBK) and hybridized for 20 h at 55 °C in an Agilent Hybridization oven (Agilent Technologies, Santa Clara, CA, USA). The hybridized microarrays were washed, and hybridized images were scanned using Agilent’s DNA microarray scanner and quantified with Feature Extraction Software (Agilent Technologies). All data were normalized (set measurements less than 0.01 to 0.01) and fold-changed probes (not less than 2.0-fold between test and control) were chosen and analyzed by using GeneSpringGX 7.3 (Agilent Technologies).

Jung J.H., Shin E.A., Kim J.H., Sim D.Y., Lee H., Park J.E., Lee H.J, & Kim S.H. (2019). NEDD9 Inhibition by miR-25-5p Activation Is Critically Involved in Co-Treatment of Melatonin- and Pterostilbene-Induced Apoptosis in Colorectal Cancer Cells. Cancers, 11(11), 1684.

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Gene Expression Analysis of Cytokines and Receptors

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Gene expression analysis was performed using topic-defined PIQOR Cytokine & Receptors Microarrays by MACSmolecular Genomics Services (Miltenyi Biotec GmbH). Briefly, RNA was isolated using standard RNA extraction protocols (NucleoSpin RNA II; Macherey-Nagel). Integrity of total RNA was evaluated using the Agilent Bioanalyzer 2100 system (Agilent Technologies). RNA integrity number was between 6.7 and 7.8, and thus, samples were considered suitable for further processing. 1 μg of each total RNA sample was used for the linear T7-based amplification step and amplified RNA checked with Agilent Bioanalyzer 2100 system. Samples were then labeled according to the PIQOR User Manual, and fluorescently labeled samples were hybridized overnight to topic-defined PIQOR Cytokine & Receptors Microarrays Mouse Antisense using the a-Hyb Hybridization Station. Fluorescent signals of the hybridized PIQOR Microarrays were detected using Agilent’s DNA microarray scanner (Agilent Technologies). Mean signals and mean local background intensities were obtained for each spot of the microarray images using the ImaGene software (Biodiscovery). Low-quality spots were flagged and excluded from data analysis. Unflagged spots were analyzed with the PIQOR Analyzer software.

Renga G., Bellet M.M., Pariano M., Gargaro M., Stincardini C., D’Onofrio F., Mosci P., Brancorsini S., Bartoli A., Goldstein A.L., Garaci E., Romani L, & Costantini C. (2020). Thymosin α1 protects from CTLA-4 intestinal immunopathology. Life Science Alliance, 3(10), e202000662.

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