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The methylated DNA immunoprecipitation microarray (MeDIP-chip) is a genome-wide, high-resolution approach to detect DNA methylation in whole genome or CpG (cytosine base followed by a guanine base) islands. The method utilizes anti-methylcytosine antibody to immunoprecipitate DNA that contains highly methylated CpG sites. Enriched methylated DNA can be interrogated using DNA microarrays or by massive parallel sequencing techniques. This combined approach allows researchers to rapidly identify methylated regions in a genome-wide manner, and compare DNA methylation patterns between two samples with diversely different DNA methylation status. MeDIP-chip has been applied successfully for analyses of methylated DNA in the different targets including animal and plant tissues (1, 2). Here we present a MeDIP-chip protocol that is routinely used in our laboratory, illustrated with specific examples from MeDIP-chip analysis of breast cancer cell lines. Potential technical pitfalls and solutions are also provided to serve as workflow guidelines.
Epigenetic modification involves DNA methylation, covalent modification of histones and small inhibitory RNA molecules known as microRNAs (miRNAs) (3). DNA methylation is a heritable, enzyme-induced modification without changing the nucleotide sequence of the DNA base pairs. DNA methylation involves transfer of a methyl-group to the 5-carbon on the cytosine in a CpG dinucleotide via DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) (4). Most of the CpG dinucleotides are unevenly distributed within the genome and regions high in these dinucleotides termed CpG islands. The haploid human genome consists of 29,848,753 CpGs and about 6% of all CpGs are located in the CpG islands (5). These islands are frequently located in the 5′-untranslated region and the first exon of approximately 60% of all genes. Methylation of CpG islands affects the transcriptional activation of genes. It is generally accepted that a high level of promoter CpG island methylation results in gene silencing (6). In the normal genome, DNA methylation is essential for proper development, chromosomal integrity, maintenance of gene expression states, and X chromosome inactivation (7, 8). In primary human tumors, methylation patterns are severely altered. This includes hypermethylation of CpG islands and genome-wide hypomethylation (9, 10). Because DNA methylation has significant effects on gene function and expression, detection of DNA methylation becomes an active area of research for the understanding of normal biological processes and tumorigenesis.
There are several methods available for the determination of methylation patterns and the quantitative assessment of methylation levels in sample tissues (11). These include methods for interrogating the combined methylation status of several CpG sites in a single gene (methylation-specific polymerase chain reaction or MSP and MethyLight; 12, 13), methods for interrogating methylation status of individual CpG sites present in a gene (combined bisulfite restriction analysis or COBRA and methylation-specific single-nucleotide primer extension or MS-SNuPE; 14, 15), methods for interrogating multiple CpG loci in many genes (methylation-specific oligonucleotide microarray or MSO microarray; 16), and methods for high-throughput, genome-wide epigenetic analysis (differential methylation hybridization or DMH; 17).
A large number of currently available techniques used for studying global methylation differences involve the use of methylation sensitive restriction enzyme(s) thereby limiting these approaches to profile genomic regions containing these restriction site motifs. In this regard, the methylated DNA immunoprecipitation (MeDIP)-chip approach is less biased and therefore more appealing (2, 18). The general strategy for the MeDIP-chip procedure is outlined in Fig. 10.1. Genomic DNA is sheared to low molecular weight fragments (average 400 bp) (see Note 1). Methylated DNAs are immunoprecipitated with the anti-methyl-cytosine antibody, and may be PCR-amplified if source material is limited (see Note 2). Input and methylated DNA can be subsequently labeled with fluorescent dyes Cy3 (green) and Cy5 (red), pooled, denatured, and hybridized to a microarray slide containing all the annotated human CpG islands (n = 27,800) or other whole genome or promoter microarray designs (see Note 3). The slide is scanned using a GenePix 4000B scanner and each image is analyzed with the GenePix Pro 6.0 image analysis software. MeDIP-chip has proven to be an efficient and robust method for analyzing DNA methylation at a genome-wide scale. Recently, several different companies have provided array designs to fit customer needs including whole-genome survey sets and promoter sets so that you can obtain comprehensive data from your methylation samples.
1Different researchers have suggested widely varying values from 200 to 1000 bp. For example, a study reported a fragment size of 600 bp (20) whereas another reported a lower fragment size of 300–600 bp (21) in their MeDIP protocol. In the protocol listed by a commercial company (NimbleGen System, Inc) a much wider fragment size range (200–1000 bp) is cited. The protocols of chromatin immunoprecipitation (ChIP) are very similar to MeDIP. The average fragment size after sonication is around 200–600 bp (21, 22).
2The amount of immunoprecipitated DNA is typically very small. One solution is to perform multiple pull down experiments. If source material is limited, one can amplify the pull down DNA prior to labeling and microarray analysis. There are several approaches described in the literature for this step, they include whole genome amplification using the Sigma WGA2 kit, ligation-mediated PCR (LM-PCR; http://www.chiponchip.org/protocol_itm3.html) or T7 amplication (23). After amplifying MeDIP and input samples, the resultant amplicons should be evaluated on an agarose gel to validate that their fragment size range matches the pull down DNA and input DNA. It is also pertinent that there should not be distinct banding pattern present in the amplified products.
|Company||Array designs||Total probes||Probe length|
|NimbleGen||Whole-Genome||2.1 million||50– mer|
|CpG island-Plus-Promoter||385,000||50–75 mer|
|Agilent Technologies||Human CpG island||237,220||95 bp|
|Mouse CpG island||97,652||95 bp|
|Phalanx Biotech||Human One Array||32050||60 mer|
|Mouse One Array||31802||71 Mer|
4There are several genomic DNA isolation kits commercial available now, you can choose one of them. Purity is determined by calculating the ratio of absorbance at 260 nm to absorbance at 280 nm. Pure DNA has an A260/A280 ratio of 1.8–2.0 indicating the absence of protein and an A260/230 ratio of >2.0, indicating the absence of other organic compounds such as ethanol. Impure DNA will lead to nonspecific binding and affect MeDIP pull down.
5After isolation of genomic DNA from your samples, run a 1.5% agarose gel to check the quality of the DNA and make sure there is no contamination with RNA, since the antibody also can recognize 5-methylcytidine in RNA. If there is a contamination with RNA, you may see smear RNA located in the front of the gel.
6Wide range of genomic DNA can be successfully fragmented by sonication (from breast progenitor cells to breast cancer cell lines). However, the efficiency of sonication varies with DNA concentration, and machine itself (probe-type sonicator produces different outcomes in comparison to Bioruptor). Therefore, it is very important to systematically check the size of the fragmented DNA. Some of the factors that will alter the fragment size such as water temperature, ice/water ratio in the sonication vessel, DNA dissolved in the water or IP buffer, duration of reset between each energy pulse, DNA concentration and volume of DNA per tube, and batch of the eppendorf tubes, etc.
7MeDIP-chip is only limited to identify CpG islands that are highly methylated in genomes. This is because the antibody used in the assay can only bind to >4 nearby methylated CpG sites. Therefore, low-density methylation of CpG islands is likely not detectable by MeDIP-chip. To improve the methylation coverage by MeDIP-chip, methyl-CpG immunoprecipitation (MCIp, 24) assay is one of the alternative methods.
8There are several different 5-methylcytidine antibodies available now.
|33D3||Mouse||Monoclonal Ab||GenWay||1 mg/ml|
|33D3||Mouse||Monoclonal Ab||Eurogentec||1 mg/ml|
|33D3||Mouse||Monoclonal Ab||ProSci||0.05 mg|
|33D3||Mouse||Monoclonal Ab||Epigentek||1 mg/ml|
|33D3||Mouse||Monoclonal Ab||Affinity BioReagents||100 μg|
|33D3||Mouse||Monoclonal Ab||Santa Cruze||50 μg/0.5 ml|
|33D3||Mouse||Monoclonal Ab||AbCam||50 μg|
|33D3||Mouse||Monoclonal Ab||AbD SeroTec||0.1 mg|
9NanoDrop ND-3300 Fluorospectrometer uses PicoGreen dye to stain nucleic acid for quantitating double-stranded DNA (dsDNA). The PicoGreen assay provides a highly sensitive means of dsDNA quantiation with minimal consumption of sample. The ND-3300 fluorospectrometer has demonstrated a detection range for dsDNA bound with PicoGreen reagent of 1 –1000 pg/μml. Compared to the NanoDrop ND-1000, ND-3300 provides more accurate readout especially for the immunoprecipitation DNA samples.
10In order to evaluate the enrichment of methylated DNA after MeDIP, we use quantitative PCR to measure the change of Ct between immunoprecipitation DNA and input DNA. We usually can have 40–200-fold increase after MeDIP based on different target genes.