Genome-wide DNA-M was measured at the University of Minnesota Genomics Center via Illumina Infinium Human Methylation450K BeadArray (Illumina) and the full QA/QC and analytical procedures described elsewhere [37 (link)]. Briefly, samples were randomized across multiple batches, stratified by birthweight group and gender. Arrays were processed and normalized using standard methods; poorly-detected probes, probes measuring DNA-M at X- and Y-linked loci, and SNP-associated loci were excluded [38 (link)], and data were standardized across batches to remove technical variations [39 (link)]. DNA-M data were analyzed as β-values, which can be interpreted as the proportion of methylated alleles for that individual CpG site. DNA-M array data for the RICHS placenta are available via the NCBI Gene Expression Omnibus (GEO) accession number GSE75248; we provide a supplemental file herein which includes log-Cd, log-PCDHAC1, and a variable for cross-linking these data (Supplemental Data ).
Infinium human methylation450k beadarray
Manufactured by Illumina
Sourced in United States
The Infinium Human Methylation450K BeadArray is a microarray-based platform designed for the analysis of DNA methylation. It provides a comprehensive coverage of CpG sites across the human genome, enabling researchers to investigate epigenetic modifications associated with various biological processes and disease states.
Automatically generated - may contain errors
3 protocols using infinium human methylation450k beadarray
1
DNA Methylation Profiling of Placenta
2
DNA Methylation Profiling of BRD1 Gene
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Two genomic regions upstream of the BRD1 gene were selected for pyrosequencing and amplified with the primers listed in S3 Table . The first assay (Pyrosequencing region 2) was designed to target rs138880 and cg15145965 (specifying a probe for a specific CpG site on the Illumina Infinium Human Methylation 450K Bead Array). The second assay (Pyrosequencing region 3) was designed to target cg06057569 and CpG sites in its close proximity. As described above, the primers were designed to be methylation independent and the highest possible annealing temperature was used. Amplification was performed as described above with the following deviations: 50 μL reactions were prepared with 40 ng bisulfite converted DNA (theoretical amount) and a final concentration of 0.2 μM of each primer was used. The annealing temperature for each assay is listed in S3 Table . Amplification products were sequenced on a PyroMark Q24 Advanced (Qiagen) using the PyroMark Q24 Advanced CpG Reagents (Qiagen), according to the manufacturer’s instructions.
3
Quantification of Hydroxymethylated DNA
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The global 5-hmc was estimated using MethylFlash™ Hydroxymethylated DNA Quantification Kit (EpiGentek, USA) according to manufacturer's instruction. The 5-hmC was estimated in 200 ng of DNA using the capture and detection antibodies followed by colorimetric detection of absorbance at 450 nm using Varioskan™ Flash Multimode Reader (Thermo Fisher Scientific, USA). The amount of hydroxymethylated DNA is calculated using the following formula according to the manufacturer's instruction The clinical information and detailed description of the dataset are available at ArrayExpress. The cervical SCC and endocervical adenocarcinoma (CESC) dataset consisting of DNA methylation data (n = 260; Illumina Infinium Human methylation 450 K bead array, Illumina, USA) and RNAseq data (n = 256) were available at the time of our analysis. A β value ≥0.2 was considered as methylated. The β value was considered as significantly hypermethylated only if the value was found in more than 5% of tumors, as published earlier. 30
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