Genechip hybridization oven 640

Manufactured by Thermo Fisher Scientific

Sourced in United States

The GeneChip Hybridization Oven 640 is a laboratory equipment designed for the hybridization of DNA microarray samples. It provides a controlled environment for incubating and mixing samples during the hybridization process.

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55 protocols using genechip hybridization oven 640

Genome-Wide SNP Genotyping and CNV Analysis

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Fifty nanograms of genomic DNA was used for a genotyping array using Genome-Wide Human SNP Array 6.0 (ThermoFisher Scientific, Waltham, Massachusetts, USA) in accordance with the manufacturer’s instructions. This product contains approximately 1.8 million genetic markers that cover the whole genome, including more than 906 600 SNPs and more than 946 000 probes for the detection of CNVs. Genomic DNA (250 ng) was digested with Sty and Nsp. Fragmented and labeled DNA was hybridized at 50 °C and 60 rpm for 16 h using GeneChip Hybridization Oven 640 (Thermo Fisher Scientific). After washing and staining with GeneChip Fluidics Station 450 (Thermo Fisher Scientific), the array cartridges were scanned with a GeneChip Scanner 3000 7G System. After scanning the array cartridge, quality check was performed by Genotyping Console software. We ascertained whether all the array data fulfilled the following criteria: quality check >0.4 and median of the absolute values of all pairwise differences (MAPD) <0.4, as recommended quality check metrics according to the manufacturer’s instructions. Some array data were excluded from CNV analysis because they did not fulfill the criteria. All of the DNA microarray data that fulfilled the criteria were deposited at Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE144918.

Sakamoto Y., Shimoyama S., Furukawa T., Adachi M., Takahashi M., Mikami T., Kuribayashi M., Osato A., Tsushima D., Saito M., Ueno S, & Nakamura K. (2021). Copy number variations in Japanese children with autism spectrum disorder. Psychiatric Genetics, 31(3), 79-87.

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Extracellular Vesicle miRNA Profiling

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Initially, two total RNA extractions from EVs were performed using the miRNeasy Mini Kit according to the manufacturer’s protocol (Qiagen GmbH, Hilden, Germany). The concentration and purity of RNA were assessed using spectrophotometry (NanoDrop ND-1000; Thermo Fisher Scientific, Wilmington, DE, USA), and RNA integrity was checked using a 2100 Bioanalyzer (Agilent Genomics, Santa Clara, CA, USA) before microarray hybridization. The samples were hybridized, washed, and stained using a GeneChip Hybridization Oven 640, GeneChip Fluidics Station 450 (Affymetrix; Thermo Fisher Scientific Inc., Waltham, MA, USA), and Affymetrix GeneChip^® miRNA 4.0 (Affymetrix; Thermo Fisher Scientific Inc.) using the protocol provided by Affymetrix. The GeneChip miRNA 4.0 array contains over 30,000 probes from Sanger miRBase v.20. The array was analyzed using the Transcriptome Viewer software (KURABO, Tokyo, Japan). The miRNA signal values were standardized by global normalization (log transformation of data and median alignment). Relative expression levels for each miRNA were validated by one-way analysis of variance or t-test (p < 0.05). MicroRNAs whose expression levels showed at least a 2-fold difference (|log2-fold change| > 1 and p < 0.05) between the test and control samples were analyzed.

Maruoka H., Tanaka T., Murakami H., Tsuchihashi H., Toji A., Nunode M., Daimon A., Miyamoto S., Nishie R., Ueda S., Hashida S., Terada S., Konishi H., Kogata Y., Taniguchi K., Komura K, & Ohmichi M. (2022). Cancer-Specific miRNAs Extracted from Tissue-Exudative Extracellular Vesicles in Ovarian Clear Cell Carcinoma. International Journal of Molecular Sciences, 23(24), 15715.

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Transcriptome Analysis of Nerve Regeneration

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Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA, USA) from the proximal and distal nerve stumps according to the manufacturer's protocols. RNA quality, amplification, labeling and hybridization of each sample were carried out using the Affymetrix GeneChip Hybridization Oven 640 and Gene Array Scanner 3000 (Affymetrix, Santa Clara, CA, USA). The arrays were scanned and the subsequent data compiled using GeneChip Scan Control software (Affymetrix). All steps, from RNA amplification to final scanner output, were conducted by the National Engineering Center for Biochip, Shanghai, China. Three biological replicates were performed at each time point. Differentially-expressed genes were identified by GeneChip scanner at each time point, with absorbance ratios set for different genes.

Jiang N., Li H., Sun Y., Yin D., Zhao Q., Cui S, & Yao D. (2014). Differential gene expression in proximal and distal nerve segments of rats with sciatic nerve injury during Wallerian degeneration. Neural Regeneration Research, 9(12), 1186-1194.

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Pooled Multiplex PCR Microarray Analysis

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The products from five multiplex PCR assays were pooled together and subjected to purification (Qiagen, Germany), processing, and hybridization in a GeneChip Hybridization Oven 640 (Affymetrix Inc, Santa Clara, CA) according to the manufacture's recommended protocol(Malanoski et al., 2006 (link); Lin et al., 2007 (link)). However, the hybridization was performed at 49°C for 12 h, which was much shorter than the time previously reported resequencing microarray adopted(Lin et al., 2007 (link), 2009 (link)). The process of staining and scanning followed by hybridization were performed in a GeneChip Fluidics Station 450 (Affymetrix Inc, Santa Clara, CA). The data collected by microarray were transferred to a computer to create scanned images. A FASTA output file produced by processing the scanned image using TessArray Sequence Analysis (TSEQ) (Malanoski et al., 2006 (link)) was subjected to alignment with corresponding detector tile sequences. The alignment results were evaluated by “C3 Score,” which means the total number of TSEQ-identified nucleotides that appear in runs of three or more consecutive (non-N) base calls, expressed as percentage of the length (nucleotides) of each RPM-IVDC1 detector tile sequence (Lin et al., 2009 (link)).

Shen H., Zhu B., Wang S., Mo H., Wang J., Li J., Zhang C., Zeng H., Guan L., Shi W., Zhang Y, & Ma X. (2015). Association of targeted multiplex PCR with resequencing microarray for the detection of multiple respiratory pathogens. Frontiers in Microbiology, 6, 532.

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Profiling miRNA Changes in ALDH+ and ALDH- Cells

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ALDH⁻ and ALDH⁺ cells isolated from DOX-untreated SUM159-shXIST cells were treated with or without (CTL) DOX at 1 µg/ml for 3 days, miRNAs from each sample were extracted using miRNeasy Mini kit (Qiagen) and analyzed by GeneChip™ miRNA 4.0 Array (ThermoFisher Scientific) according to manufacturer’s instructions. miRNA-containing total RNA (300 ng) was biotin-labeled using the FlashTag Biotin RNA Labeling kit (Afymetrix, USA) and hybridized in the GeneChip Hybridization Oven 640 (Affymetrix, USA) at 48 °C for overnight. After washed and stained in the GeneChip Fluidics Station 450 (Afymetrix, USA), arrays of different samples were scanned with a GeneChip Scanner 3000 7 G (Afymetrix, USA) and signal strength was evaluated using the Expression Console Software (EC) v1.2 (ThermoFisher Scientific). To identify differentially expressed miRNAs in ALDH⁻ and ALDH⁺ cells treated with or without DOX, acquired array data were analyzed using Multi Experiment Viewer (MeV v4.9.0; The Institute for Genomic Research) and miRNAs with an absolute value of fold change ≥ 0.6 were identified as potential miRNAs significantly changed upon DOX-induced XIST KD.

Ma Y., Zhu Y., Shang L., Qiu Y., Shen N., Wang J., Adam T., Wei W., Song Q., Li J., Wicha M.S, & Luo M. (2023). LncRNA XIST regulates breast cancer stem cells by activating proinflammatory IL-6/STAT3 signaling. Oncogene, 42(18), 1419-1437.

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Microarray Analysis of Mesenchymal Stem Cells

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MSCs cultured at 100 cells/cm² for 7 days in CCM and in hanging drops for 3 days in CCM, MesenC, StemP, or StemP HSA were harvested for total RNA using RNeasy Mini Kit with Qiashredder columns (Qiagen) and DNase digestion (Qiagen). RNA was quantified with Nanodrop spectrophotometer (Thermo Scientific) and total of 2 µg of RNA from each sample was applied for microarrays using Whole Transcript Sense Target Labeling Assay protocol (Affymetrix) according to manufacturer’s directions. Total of 5.5 µg of cDNA was fragmented, labeled, and then hybridized (GeneChip Hybridization Oven 640, Affymetrix) on to Human Exon 1.0 ST arrays (Affymetrix). Arrays were washed, stained (GeneChip Fluidics Station 450, Affymetrix) and scanned with GeneChip Scanner (Affymetrix) and the raw data files (CEL-files) were transferred into Partek Genomics Suite 6.4 (Partek). The data were normalized using robust multi-array (RMA) algorithm and gene level analysis was performed with the Partek software. Data from spheres were compared to data from standard adherent cells to obtain fold changes.

Ylostalo J.H., Bartosh T.J., Tiblow A, & Prockop D.J. (2014). Unique characteristics of human mesenchymal stem/progenitor cells (MSC) pre-activated in 3D cultures under different conditions. Cytotherapy, 16(11), 1486-1500.

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Genome-Wide SNP Profiling of PBMCs from β-Thalassemic Patients

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PBMC from β-Thalassemic patients transplanted with HLA-related HSCT and donors were genotyped with the Affymetrix Genome-Wide Human SNP 6.0 Array (Affymetrix, Inc. Santa Clara, CA, USA), including probes for more than 906,000 SNPs and for the detection of copy number variants (CNVs) ^{13 (link)}, according to manufacture’s instructions. Briefly, total genomic DNA (500 ng) was digested with Nsp I and Sty I enzymes, ligated to adaptors and amplified using a primer that recognizes the adaptor sequence. The amplified DNA was then fragmented, labelled and hybridized to oligonucleotide probes attached to the surface of an array in a GeneChip Hybridization Oven 640 (Affymetrix, Inc.), followed by washing and staining procedures performed on a GeneChip Fluidics Station 450 (Affymetrix, Inc.). Arrays were scanned using the GeneChip Scanner 3000 7G (Affymetrix, Inc.).

Piras I.S., Angius A., Andreani M., Testi M., Lucarelli G., Floris M., Marktel S., Ciceri F., La Nasa G., Fleischhauer K., Roncarolo M.G., Bulfone A., Gregori S, & Bacchetta R. (2014). BAT2 and BAT3 polymorphisms as novel genetic risk factors for rejection after HLA-related stem cell transplantation. Bone marrow transplantation, 49(11), 1400-1404.

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Genome-Wide SNP Profiling of PBMCs from β-Thalassemic Patients

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Transcriptome profiling of progenitor cells

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Progenitor cells or their progeny from in vitro culture were sorted as described above. RNA from sorted populations was extracted with a NucleoSpin RNA XS Kit (Machery-Nagel), then was amplified with WT Pico System (Affymetrix) and hybridized to GeneChip Mouse Gene 1.0 ST microarrays (Affymetrix) for 18 h at 45 °C in a GeneChip Hybridization Oven 640. The data was analyzed with the Affymetrix GeneChip Command Console. Microarray expression data was processed using Command Console (Affymetrix, Inc) and the raw (.CEL) files generated were analyzed using Expression Console software with Affymetrix default RMA Gene analysis settings.(Affymetrix, Inc). Probe summarization (Robust Multichip Analysis, RMA), quality control analysis, and probe annotation were performed according to recommended guidelines (Expression Console Software, Affymetrix, Inc.). Data were normalized by robust multiarray average summarization and underwent quartile normalization with ArrayStar software (DNASTAR).

Durai V., Bagadia P., Granja J.M., Satpathy A.T., Kulkarni D.H., Davidson JT I.V., Wu R., Patel S.J., Iwata A., Liu T., Huang X., Briseño C.G., Grajales-Reyes G.E., Wöhner M., Tagoh H., Kee B.L., Newberry R.D., Busslinger M., Chang H.Y., Murphy T.L, & Murphy K.M. (2019). Cryptic activation of an Irf8 enhancer governs cDC1 fate specification. Nature immunology, 20(9), 1161-1173.

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Mouse Gene Expression Microarray Analysis

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RNA was extracted with a RNAqueous-Micro Kit (Ambion) or a NucleoSpin RNA XS Kit (Machery-Nagel), then was amplified with Ovation Pico WTA System (NuGEN) or WT Pico System (Affymetrix) and hybridized to GeneChip Mouse Gene 1.0 ST microarrays (Affymetrix) for 18 h at 45 °C in a GeneChip Hybridization Oven 640. The data was analyzed with the Affymetrix GeneChip Command Console. Microarray expression data was processed using Command Console (Affymetrix, Inc) and the raw (.CEL) files generated were analyzed using Expression Console software with Affymetrix default RMA Gene analysis settings (Affymetrix, Inc). Probe summarization (Robust Multichip Analysis, RMA), quality control analysis, and probe annotation were performed according to recommended guidelines (Expression Console software, Affymetrix, Inc.). Data were normalized by robust multiarray average summarization and underwent quartile normalization with ArrayStar software (DNASTAR). Unsupervised hierarchical clustering of differentially expressed genes was computed with ArrayStar (DNASTAR) with the Euclidean distance metric and centroid linkage method.

Bagadia P., Huang X., Liu T., Durai V., Grajales-Reyes G.E., Nitschke M., Modrusan Z., Granja J.M., Satpathy A.T., Briseño C.G., Gargaro M., Iwata A., Kim S., Chang H.Y., Shaw A.S., Murphy T.L, & Murphy K.M. (2019). An Nfil3–Zeb2–Id2 pathway imposes Irf8 enhancer switching during cDC1 development. Nature immunology, 20(9), 1174-1185.

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