The Illumina Infinium HumanMethylation450 BeadChip array (Illumina, San Diego, CA, USA) is widely used in various fields to measure DNA methylation. The array assessed more than 450,000 CpGs, and the coverage ratio of RefSeq genes is 99%. Thus, we used the Illumina Infinium HumanMethylation450 BeadChip array to perform genome-wide methylation analysis in 26 paired CRC tissues and adjacent noncancerous tissues. Based on the manufacturer’s instructions, the EpiTect Fast DNA Bisulfite Kit (Qiagen, Cat. no. 59826) was used to perform bisulfite conversion for 500 ng of genomic DNA per time. After calculating the sum of the methylated ratio, the methylation level of each CpG site was marked as “beta” for values ranging from 0 (unmethylated) to 1 (fully methylated).
Infinium humanmethylation450 beadchip array
Manufactured by Illumina
Sourced in United States, United Kingdom
The Infinium HumanMethylation450 BeadChip array is a high-throughput laboratory equipment used for the analysis of DNA methylation patterns across the human genome. It provides a comprehensive coverage of CpG sites, allowing researchers to assess the methylation status of over 450,000 individual cytosine-phosphate-guanine (CpG) sites.
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Lab products found in correlation
Ez dna methylation kit, by Zymo Research (21 mentions)
Ez 96 dna methylation kit, by Zymo Research (6 mentions)
Ez dna methylation gold kit, by Zymo Research (5 mentions)
Epitect fast dna bisulfite kit, by Qiagen (4 mentions)
Genomestudio, by Illumina (4 mentions)
Iscan system, by Illumina (3 mentions)
Dneasy blood and tissue kit, by Qiagen (3 mentions)
Dneasy blood tissue kit, by Qiagen (3 mentions)
Genomestudio software, by Illumina (3 mentions)
Infinium humanmethylation450 beadchip, by Illumina (2 mentions)
Zymo ez dna methylation kit, by Zymo Research (2 mentions)
Hiseq 2000, by Illumina (2 mentions)
Infinium methylationepic kit, by Illumina (2 mentions)
Qiaamp dna mini kit, by Qiagen (2 mentions)
Qiamp dna mini kit, by Qiagen (2 mentions)
Infinium ffpe dna restore kit, by Illumina (2 mentions)
Nanodroptm 8000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Humanht 12 beadchip, by Illumina (2 mentions)
Qiaamp dna blood mini kit, by Qiagen (2 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (2 mentions)
125 protocols using infinium humanmethylation450 beadchip array
1
Genome-wide DNA Methylation Analysis of Colorectal Cancer
2
DNA Methylation Analysis Using Illumina 450K
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Samples were analyzed for genome-wide DNA methylation patterns using the Illumina Infinium HumanMethylation450 BeadChip array (Illumina Inc., CA, USA). Genomic DNA was extracted from 250 μL of whole blood (drawn on EDTA tubes), using the QIAamp 96 DNA Blood Kit (QIAGEN, Hilden, Germany). One microgram of DNA was bisulfite converted, using the EZ DNA Methylation Kit (Zymo Research Corporation, Orange, CA, USA) according to manufacturer’s instructions. After bisulfite conversion, DNA was whole-genome amplified, enzymatically fragmented, and hybridized to the Illumina Infinium HumanMethylation450 BeadChips (Illumina Inc., CA, USA), according to the manufacturer’s protocols. Samples from the different groups (Italy and Poland, T0 and T1) were accurately randomized across the experimental sessions. Arrays were scanned using the HiScan instrument (Illumina Inc., CA, USA). Raw fluorescence intensities were extracted using minfi Bioconductor package, and normalization was performed using the preprocessQuantile function (Touleimat and Tost 2012 (link)).
3
Genome-wide DNA Methylation Profiling
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Genome-wide methylation in peripheral blood cells from a subset of the EIRA, EIMS and InCHIANTI cohorts were evaluated by Illumina Infinium Human Methylation 450 BeadChip according to the manufacturer’s recommendations. Illumina Infinium Human Methylation 450 BeadChip array quantifies methylation levels at specific loci within the genome. The percentage methylation value for a particular CpG site, which represents the fraction of DNA with this CpG site methylated, was calculated on a scale of 0–1, per Illumina’s recommendations, using the formula: where M and U represent the methylated and unmethylated signal intensities, respectively. Methylation data from EIRA was published previously [12 (link)] and can be downloaded from Gene Expression Omnibus [GEO:GSE42861].
4
DNA Methylation Profiling of Chondrogenesis
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Genomic DNA was extracted from cells prior to the induction of chondrogenesis (Day0) and from Day14 cartilagenous discs by disruption in Invitrogen PureLink Genomic Digestion Buffer (Life Technologies, Paisley, UK) using a small disposable plastic pestle and an aliquot of Molecular Grinding Resin (G-Biosciences, St. Louis, MO) followed by proteinase K digestion for 1 hour at 37 °C then nucleic acid purification with Invitrogen PureLink Genomic DNA Kit according to manufacturer’s instructions (Life Technologies). 1 µg of genomic DNA was bisulphite converted using the EpiTect Bisulfite Kit (Qiagen, Manchester, UK). DNA methylation profiling of the samples was carried out by Cambridge Genomic Services (Cambridge, UK), using the Illumina Infinium HumanMethylation450 Beadchip array (Illumina Inc., San Diego, USA).
5
Integrative Molecular Profiling of LUAD
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RNA-sequencing (RNA-seq) data, DNA methylation data generated by Illumina Infinium HumanMethylation 450 BeadChip array, copy number variation (CNV) data (Masked Copy Number Segment, affymetrix snp 6.0), single nucleotide variant (SNV) data (MuTect2. Variant0. Maf), and LUAD clinical follow-up pathological data were downloaded from The Cancer Genome Atlas (TCGA) (https://portal.gdc.cancer.gov/ ). The expression profile data was changed from fragments per kilobase million (FPKM) to transcripts per million (TPM) and transformed into log2. The processing of DNA methylation data involved the use of the k-nearest neighbors (KNN) algorithm to complete the missing values and retrograde, the removal of cytosine-guanine (CpG) sites with cross-reflection, and the exclusion of methylation sites on X and Y chromosomes. The stemness index was derived from Malta et al. (13 (link)). messenger RNA (mRNA) expression-based stemness indices (mRNAsi) was calculated based on the expression profile data, while DNA methylation-based stemness index (mDNAsi) was calculated based on the methylation data. The immune cell biomarkers gene set was obtained from the article of Charoentong et al. (14 (link)), which includes 782 genes. The workflow of this study is summarized in Figure S1 . The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013).
6
Functional Analysis of Orofacial Cleft DMRs
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To explore the function of any OFC-associated DMRs, we used the missMethyl [35 (link)] R package to test for enrichment of any gene ontology (GO) classification terms or Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathways. This method corrects for biases in the genomic coverage of the Illumina Infinium HumanMethylation450 BeadChip array. We also looked up gene annotations from our DMRs in recently curated lists of OFC-related genes from (1) the DisGeNET database of diseases and related genes from human, rat and mouse studies [36 (link)] and (2) a bioinformatics study of OFC-related genes in human and animal studies published by Funato et al. [37 (link)].
7
DNA Methylation Profiling via Infinium 450K
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DNA samples were bisulfate-converted using a ZymoEZ DNA Methylation Kit (Zymo Research). After being amplified and enzymatically fragmented, the resulted fragments were purified by isopropanol precipitation and hybridized to the Infinium Human Methylation 450 BeadChip array (Illumina Inc., San Diego, CA, USA). After hybridization for 18 hours, extension, staining, and washing were performed successively. Finally, the BeadChip was imaged using the iScan system (Illumina, Inc.). In total, over 485,000 methylation sites were analyzed per sample, and the entire assay was performed according to the manufacturer’s instructions.
8
Probe Set Annotation for Illumina HumanMethylation450 Beadchip
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For probe set annotation in the Illumina Infinium HumanMethylation450 Beadchip array, we used the expanded annotation table provided by Price et al. [28 (link)]. This file was used for associating gene and transcript with respective CpG site, the distance to the closest transcription start site and the occurrence of any single nucleotide polymorphism (SNP) loci in the CpG site. CpG sites were excluded from the analysis if carrying a known SNP locus or if located outside the area 1500 bp upstream or downstream from the transcriptional start site (TSS1500 interval). The probe set annotation file provided by Affymetrix (Affymetrix Inc., Santa Clara, CA) was used to associate a gene transcript with probes on the Affymetrix chip. On this array, probes may be annotated to more than one individual transcript.
9
Illumina DNA Methylation Profiling
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DNA methylation profiles were assessed by the Illumina Infinium Human Methylation 450 Beadchip array (Illumina, San Diego, CA, USA). As previously described [50 (link)], samples were analyzed following the manufacturer's instruction at the Genomics and Proteomics Core Facility of the German Cancer Research Center, Heidelberg, Germany. Illumina'sGenomeStudio® (version 2011.1; Illumina.Inc .) was employed to extract DNA methylation signals from the scanned arrays (Module version 1.9.0; Illumina.Inc .). The methylation status of a specific CpG site was quantified as a β value ranging from 0 (no methylation) to 1 (full methylation). According to the manufacturer's protocol, no background correction was done and data were normalized to internal controls provided by the manufacturer. All controls were checked for inconsistencies in each measured plate. Signals of probes with a detection p-value > 0.05 were excluded from analysis. We used the Illumina normalization and preprocessing method implemented in Illumina's Genomestudio (“Illumina normalization”). In addition, as previously described [51 (link)], we measured the cotinine levels in serum samples of the discovery panel, using the customized version of an enzyme-linked immunosorbent assay (Inspec II-Cotinine-EIA; Mahsan Diagnostika).
10
Methylation Analysis of Eosinophils in Asthma
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We measured DNA methylation levels on a subset of individuals from the SLSJ asthma cohort using whole blood (167 samples) and isolated eosinophils from blood (24 samples). Eosinophil cells isolation was performed as described in Ferland et al. [36 (link)] and DNA extraction and sodium bisulfite conversion were described in Liang et al. [37 (link)]. Methylation levels were assessed using the Infinium HumanMethylation450 BeadChip array (Illumina, San Diego, CA, USA). Normalization steps were described in Morin et al. [38 (link)].
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