Genetitan multi channel instrument

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

The GeneTitan Multi-Channel instrument is a high-throughput microarray platform designed for gene expression and genotyping analysis. The core function of the instrument is to automate the processing and analysis of microarray samples, enabling efficient and accurate data generation.

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GeneTitan instrument (33 citations) GeneTitan (24 citations) Multi-Channel Instrument (6 citations)

37 protocols using genetitan multi channel instrument

Microarray Analysis of Whole Mouse Genome

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Microarray assays were performed by Edinburgh Genomics, University of Edinburgh (https://genomics.ed.ac.uk/). Total RNA was labelled using the IVT Express Kit (Affymetrix). First-strand cDNA was synthesised and converted to double-stranded DNA template for transcription and synthesis of aRNA incorporating a biotin-conjugated nucleotide. aRNA was purified and fragmented prior to hybridisation on Affymetrix arrays. Biotin-labelled aRNA was hybridized to the whole mouse genome HT MG-430 PM array plate (Affymetrix, CA, USA) representing >39,000 transcripts, using the GeneTitan multi-channel instrument (Affymetrix).

Grabert K., Michoel T., Karavolos M.H., Clohisey S., Baillie J.K., Stevens M.P., Freeman T.C., Summers K.M, & McColl B.W. (2016). Microglial brain region-dependent diversity and selective regional sensitivities to ageing. Nature neuroscience, 19(3), 504-516.

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Genome-Wide Genotyping with Thermo Chip

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In this study, we selcted Thermo Scientific Genotyping Chip (Thermo Fisher Scientific Inc., Waltham, MA, USA), and using Gene Titan multi-channel instrument (Affymetrix, Inc., Santa Clara, CA, USA) and Axiom Analysis Suite 6.0 software (Thermo Fisher Scientific Inc.) for genotyping. Within the scope of our study subjects, we conducted a genome-wide scan through Axiom and the results showed that it contained a total of 819,009 million loci. After excluding insertion-deletion, copy number variation, and duplication, 756,558 loci remain. These loci meet the following conditions: sample call rate >0.95, maker call rate >0.90 and Hardy–Weinberg equilibrium (HWE) >5×10⁻⁶.

Wang F., Luo D., Chen J., Pan C., Wang Z., Fu H., Xu J., Yang M., Mo S., Zhuang L., Ye L, & Wang W. (2022). Genome-Wide Association Analysis to Search for New Loci Associated with Lifelong Premature Ejaculation Risk in Chinese Male Han Population. The World Journal of Men's Health, 40(2), 330-339.

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Genotyping of Arachis NAM Populations

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DNA samples from all the NAM lines used in this study were extracted from young leaves using Thermo Scientific GeneJET Plant Genomic DNA Purification Mini Kit. The DNA samples were checked for quality on 0.8% agarose gels and quantified on a Nanodrop 8000 Spectrophotometer (Thermo Scientific, Pittsburgh, PA). Affymetrix GeneTitan platform was used to genotype both NAM populations with the 58K SNP ‘Axiom_Arachis’ array (Clevenger et al., 2017; Pandey et al., 2017b). Initially, the target probes for 581 samples for NAM‐T and 496 samples for NAM‐F were prepared using a minimum of 20 μL DNA with a concentration 10 ng/μL. The samples were then amplified, fragmented and hybridized on the array chip followed by single‐base extension through DNA ligation and signal amplification according to the procedure explained in the Affymetrix Axiom® 2.0 Assay Manual (http://axiom_2_assay_auto_workflow_user_guide.pdf). The GeneTitan Multi‐Channel Instrument (Affymetrix, Santa Clara, CA, USA) was then used for staining and scanning the samples to derive the genotypic information for each line. The genotypic data for each line were generated and stored in the form of.CEL file format.

Gangurde S.S., Wang H., Yaduru S., Pandey M.K., Fountain J.C., Chu Y., Isleib T., Holbrook C.C., Xavier A., Culbreath A.K., Ozias‐Akins P., Varshney R.K, & Guo B. (2019). Nested‐association mapping (NAM)‐based genetic dissection uncovers candidate genes for seed and pod weights in peanut (Arachis hypogaea). Plant Biotechnology Journal, 18(6), 1457-1471.

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Pigeonpea Genotyping Using Affymetrix Arrays

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Pigeonpea genomic DNA was isolated from young seedlings using CTAB method³⁸, quality checked by electrophoresis in 1% agarose gel and quantified using Nano drop spectrophotometer. For target probe preparation, 20 μl of genomic DNA with concentration of 10 ng/ul was used according to Affymetrix Axiom® 2.0 Assay Manual. The samples were amplified using Target Prep Protocol QSCB1 (P/N 702990), fragmented, hybridized on the chip followed by single-base extension through DNA ligation and signal amplification. Affymetrix GeneTitan® Multi-Channel Instrument was used for staining, washing and scanning of the chip signals as per the manufacturer’s protocol (http://media.affymetrix.com/support/downloads/manuals/axiom_2_assay_auto_workflow_user_guide.pdf). SNP allele calling was done using Axiom™ Analysis Suite version 2.0 using its three workflows i.e., Best Practices, Sample QC, Genotyping and Summary (http://media.affymetrix.com/support/downloads/manuals/axiom_analysis_suite_user_guide.pdf) on the Affymetrix Gene Titan.CEL files. The Axiom Analysis Suite requires stored library files to convert.CEL files into genotype calls. SNPs with low call rate across the samples were removed and only good quality SNPs with a DQC of >0.85 and call rates of >95% were retained for further analysis.

Singh S., Mahato A.K., Jayaswal P.K., Singh N., Dheer M., Goel P., Raje R.S., Yasin J.K., Sreevathsa R., Rai V., Gaikwad K, & Singh N.K. (2020). A 62K genic-SNP chip array for genetic studies and breeding applications in pigeonpea (Cajanus cajan L. Millsp.). Scientific Reports, 10, 4960.

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Genotyping FFPE Tissue DNA

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After the paraffin layer was removed, 1 mm diameter cores punched from FFPE tissue blocks were sectioned into 20–30 pieces using a sterile razor blade. Samples were then vortexed with 1 ml xylene, followed by 2 minutes of centrifugation at room temperature. Next, samples were again vortexed with 1 ml of 100% ethanol and pelleted by centrifugation. The supernatant was discarded and residual solvent was evaporated at room temperature. Next, DNA was purified from blood samples (Promega Wizard Genomic DNA Purification Kit) and FFPE tissues (QIAamp DNA FFPE Tissue Kit). To boost DNA yields prior to genotyping, 200 ng of input DNA from each sample was amplified (Affymetrix Axiom 2.0 Reagent Kit) via isothermal incubation at 37 °C for 48 hours. Then the sample DNA was fragmented into pieces ranging from 25 to 125 base pairs, followed by isopropanol precipitation. The Affymetrix GeneTitan Multi-Channel Instrument was used for sample genotyping.

Emami N.C., Leong L., Wan E., Van Blarigan E.L., Cooperberg M.R., Tenggara I., Carroll P.R., Chan J.M., Witte J.S, & Simko J.P. (2016). Tissue Sources for Accurate Measurement of Germline DNA Genotypes in Prostate Cancer Patients Treated with Radical Prostatectomy. The Prostate, 77(4), 425-434.

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Cell-free DNA Analysis for Aneuploidy Screening

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All sample processing from plasma was performed under a blinded protocol in Ariosa Diagnostics' certified and accredited clinical laboratory. A single sample was processed for each subject. As previously described,6, 7, 14 cfDNA was purified from each plasma sample, and DANSR products were made from nonpolymorphic assays on each of chromosomes 13, 18, and 21 and polymorphic assays from chromosomes 1–12. The DANSR products from each sample were hybridized to a custom DNA microarray from Affymetrix Inc. (Santa Clara, California), and imaged on an Affymetrix GeneTitan® Multi‐Channel instrument.14 Each patient sample was assayed on a single microarray. For analysis of the subset with matching fetal tissue, pure genomic DNA was isolated from fetal tissue; the DNA was sheared to small fragments with sonication prior to processing through the lab as a cfDNA sample.

Stokowski R., Wang E., White K., Batey A., Jacobsson B., Brar H., Balanarasimha M., Hollemon D., Sparks A., Nicolaides K, & Musci T.J. (2015). Clinical performance of non‐invasive prenatal testing (NIPT) using targeted cell‐free DNA analysis in maternal plasma with microarrays or next generation sequencing (NGS) is consistent across multiple controlled clinical studies. Prenatal Diagnosis, 35(12), 1243-1246.

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Comprehensive RNA Extraction and Analysis

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Total RNA was extracted using the Trizol reagent and further purified using a Qiagen RNeasy Mini Kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany). Microarrays were imaged on an Affymetrix GeneTitan® Multi-Channel Instrument. Each group was assayed on a single custom microarray. Microarrays were manufactured and processed in interconnected sets of 384. Next-generation sequencing data were produced on an Illumina HiSeq® 2500 (San Diego, Calif., USA) instrument according to the manufacturer’s instructions.

Qian W., Cai X., Qian Q., Wang D, & Zhang L. (2020). Angelica Sinensis Polysaccharide Suppresses Epithelial-Mesenchymal Transition and Pulmonary Fibrosis via a DANCR/AUF-1/FOXO3 Regulatory Axis. Aging and Disease, 11(1), 17-30.

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Microarray Analysis of Whole Mouse Genome

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Genetic Profiling of Brugada Syndrome

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Simple nucleotide polymorphism genotyping was performed on population‐optimized Affymetrix Axiom Genome‐Wide CEU 1 array plates following the standard manufacturer's protocol. Fluorescence intensities were quantified by using the Affymetrix GeneTitan Multi‐Channel Instrument, and primary analysis was conducted with Affymetrix Power Tools following the manufacturer's recommendations. After genotype calling, all individuals had a genotype call rate >97%. Simple nucleotide polymorphisms with a minor allele frequency (MAF) <10%, a call rate <95%, or with P<1×10⁻⁴ when testing for Hardy–Weinberg equilibrium were excluded. We used the MERLIN algorithm9 to detect chromosomal fragments cosegregating with the BrS phenotype, by testing a model of autosomal dominant pattern of inheritance with incomplete penetrance (80%). The maximal theoretical LOD (logarithm of odds) score was 0.82 for this family. The threshold for selecting shared genomic regions was set to 0.7 (P=0.05 uncorrected for multiple testing).

Portero V., Le Scouarnec S., Es‐Salah‐Lamoureux Z., Burel S., Gourraud J., Bonnaud S., Lindenbaum P., Simonet F., Violleau J., Baron E., Moreau E., Scott C., Chatel S., Loussouarn G., O'Hara T., Mabo P., Dina C., Le Marec H., Schott J., Probst V., Baró I., Marionneau C., Charpentier F, & Redon R. (2016). Dysfunction of the Voltage‐Gated K+ Channel β2 Subunit in a Familial Case of Brugada Syndrome. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 5(6), e003122.

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Rapamycin response in miR-150 transfected Jurkat cells

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Jurkat cells were grown in 4 independent cultures for several passages, transiently transfected with pre-miR-150 or control (scrambled) pre-miRNA and then treated with rapamycin or DMSO for 72 h. Total RNA was analyzed on the Agilent Bioanalyzer to confirm integrity. cDNA was synthesized and labeled using the Ambion whole transcript (WT) expression kit (Life Technologies), hybridized to 24-array HuGene 1.1ST plates and run on the GeneTitan Multi-Channel Instrument (Affymetrix). Data analysis was done in Genomics Suite (Partek). Probe intensities were normalized using the RMA method. Expression changes were calculated using 1-way ANOVA with the p-value < 0.05 as a cutoff for significance. Network and pathway enrichment analyses were performed using Ingenuity Pathway Analysis software and GO term enrichment was performed using GOrilla: http://cbl-gorilla.cs.technion.ac.il (27 (link)).

Podshivalova K., Wang E.A., Hart T, & Salomon D.R. (2018). Expression of the miR-150 tumor suppressor is restored by and synergizes with rapamycin in a human leukemia T-cell line. Leukemia research, 74, 1-9.

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