Computational protocol: Screening significantly hypermethylated genes in fetal tissues compared with maternal blood using a methylated-CpG island recovery assay-based microarray

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Protocol publication

[…] The MIRA-based microarray analysis was performed as described previously [] with minor modifications. Briefly, DNA was first separated from four pairs of matched fresh-frozen maternal peripheral blood and placental tissue samples using the standard phenol/chloroform technique. Then 2μg of the genomic DNA samples were sheared into 200–1000 bp fragments by Mse I (5′-TTAA) and purified to remove any fragments smaller than 100 bp using a MicroDNA Purification Kit (CoWin Biotech Company, Beijing, China) following the manufacturer’s instruction. Afterwards, the purified DNA fragments were used to enrich methylated DNA using a MBD kit (BioChain, Hayward, CA, USA) according to the manufacturer’s protocol. The MIRA-captured DNA segments were purified and amplified using GenomePlex Whole Genome Amplification Kit (Sigma) as per the supplier’s instruction. The products of whole genome amplification from the total input DNA without MBD enrichment and methylation-enriched DNA from each of the samples were labeled with cy3-dCTP and cy5-dCTP respectively, using Klenow enzyme (Takara, Dalian, China). The fluorescent dye labeled DNA was mixed and hybridized to Agilent human CpG island microarrays that were designed to interrogate 61,982 CpG dinucleotides covering 4,162 genes. After hybridization, the slides were washed and scanned on the Agilent microarray platform according to Agilent’s standard protocol. The data were extracted using Agilent Feature Extraction software. Following global mean normalization, faint probes with intensity <400 were discarded and excluded from the analysis. Unsupervised clustering analysis was performed using the Cluster software. Probes were considered positive for differential methylation between maternal blood and placental tissue if the fold changes in their MIRA/Input signaling ratios between the placental tissue and the maternal blood were >1.2 or <0.83 (q <0.05) using SAM (significance analysis of microarrays) []. The significant enrichment of the Gene Ontology (GO) terms associated with the hypermethylated genes was analyzed using the hypergeometric distribution in the R language software package. All microarray data have been submitted to the Gene Expression Omnibus [GEO: GSE35997]. […]

Pipeline specifications

Software tools Agilent Feature Extraction, SAM
Databases GEO
Application Gene expression microarray analysis
Diseases Genetic Diseases, Inborn