biology Review DNA Methylation Analysis: Choosing the Right Method Sergey Kurdyukov 1,* and Martyn Bullock 2 Received: 8 July 2015; Accepted: 22 December 2015; Published: 6 January 2016 Academic Editor: Melanie Ehrlich 1 Genomics Core facility, Kolling Institute of Medical Research, University of Sydney, Sydney 2065, Australia 2 Cancer Genetics Laboratory, Kolling Institute of Medical Research, University of Sydney, Sydney 2065, Australia; [email protected] * Correspondence: [email protected]; Tel.: +61-299-264-756 Abstract: In the burgeoning field of epigenetics, there are several methods available to determine the methylation status of DNA samples. However, choosing the method that is best suited to answering a particular biological question still proves to be a difficult task. This review aims to provide biologists, particularly those new to the field of epigenetics, with a simple algorithm to help guide them in the selection of the most appropriate assay to meet their research needs. First of all, we have separated all methods into two categories: those that are used for: (1) the discovery of unknown epigenetic changes; and (2) the assessment of DNA methylation within particular regulatory regions/genes of interest. The techniques are then scrutinized and ranked according to their robustness, high throughput capabilities and cost. This review includes the majority of methods available to date, but with a particular focus on commercially available kits or other simple and straightforward solutions that have proven to be useful. Keywords: DNA methylation; 5-methylcytosine; CpG islands; epigenetics; next generation sequencing 1. Introduction DNA methylation in vertebrates is characterized by the addition of a methyl or hydroxymethyl group to the C5 position of cytosine, which occurs mainly in the context of CG dinucleotides. Non-CpG methylation in a CHH and CHG context (where H = A, C or T) exist in embryonic stem cells and in plants. The aim of this review is to inform biologists studying DNA methylation of the pros and cons of the different assays currently available; allowing them to make an informed choice when deciding the technique that would best suit their research needs. Most importantly, the method of choice should deliver an unbiased answer to the biological question being asked by the researcher. However, there are several other key factors that must be considered when choosing a method for DNA methylation analysis: ‚ The aims of the study (e.g., the discovery of de novo epigenetic changes or the investigation of known methylation sites of specific genes of interest); ‚ The amount and quality of the DNA sample(s) (e.g., Formalin-Fixed Paraffin-Embedded (FFPE) versus frozen tissue-derived DNA); ‚ The sensitivity and specificity requirements of the study; ‚ The robustness and simplicity of the method; Biology 2016, 5, 3; doi:10.3390/biology5010003 www.mdpi.com/journal/biology Biology 2016, 5, 3 2 of 21 ‚ The availability of bioinformatics software for analysis and interpretation of the data; ‚ The availability of specialized equipment and reagents; ‚BiologyCost. 2016, 5, 3 2 of 21 FigureFigure1 1 provides provides aa graphicalgraphical guideguide forfor choosing the right method method for for a a specific specific project project using using a asimple simple algorithm. algorithm. The The following following subsections subsections of of the the review review will will describe describe each each method method,, as well asas highlighthighlight theirtheir prospros andand cons.cons. Furthermore,Furthermore, an example application of the proposedproposed algorithmalgorithm isis illustratedillustrated in Figure Figure 2.. Not Not all all possible possible techniques techniques that that exist exist will will be becovered covered in this in this review, review, as we as wewill willfocus focus on those on those methods methods that thatwe think we think are the are most the most robust, robust, simple simple to use to useandand readily readily available available to the to theresearch research community. community. A more A more comprehensive comprehensive review review of all of allavailable available techniques techniques has has been been written written by byOlkhov Olkhov-Mitsel-Mitsel and andBapat Bapat [1]. There [1]. There are also are several also several good review good reviewarticles that articles cover that particular cover particular methods methodsin much more in much detail more than detail is described than is described here [2–5] here. Additionally, [2–5]. Additionally, there are there specific are specificweb-based web-based forums forumsthat can that aid can in aidthe in quest the quest to find to findthe themost most suitable suitable method method for for analysis: analysis: epigenie.com epigenie.com [6] [6] andand http://www.protocol-online.orghttp://www.protocol-online.org [7] [7].. Furthermore Furthermore,, providers providers of of fee fee-for-for service analysis can be foundfound atat https://www.scienceexchange.comhttps://www.scienceexchange.com [8] [8] and and https://genohub.com https://genohub.com [9] [9. ]. 1.1 HPLC-UV 1.2 Mass spectrometry- Are based candidate 1.3 ELISA-based genes No 1) Whole known? genome methylation Bisulfite profiling conversion No Yes 1.6 LUMA Yes 1.4 PCR of LINE-1 (digest + followed by pyrosequencing 1.5 RFLP; AFLP pyrosequencing extension) 4) Digestion- based assay 3) Bisulfite followed by conversion 2) Search for PCR or qPCR differentially methylated 3.1 Bead array regions Bisulfite 3.4 Methylation- conversion specific PCR 3.5 HRM 3.2 PCR and 3.6 COLD-PCR sequencing 2.3 2.2 Microarray 2.1 Bisulfite 3.3 Pyrosequencing MSCC or bead array sequencing MSCC: Methyl-Sensitive Cut Counting LUMA: Luminometric Methylation Assay LINE: Long Interspersed Nuclear Elements ELISA: Enzyme-Linked Immunosorbent Assay AFLP: Amplified Fragment Length Polymorphism Enrichment for Enrichment for RFLP: Restriction Fragment Length Polymorphism methylated CpG rich regions HRM: High Resolution Melting regions (optional) COLD-PCR: explained in chapter 4.6 Figure 1. Algorithm for choosing a suitable method for DNA methylationmethylation analysis.analysis. Biology 2016, 5, 3 3 of 21 Biology 2016, 5, 3 3 of 21 Amount of starting material Cost per sample low high ≤ 1μg ≥ 1μg moderate DNA origin Number of samples 450K (Illumina) human mouse or rat other ≤ 10 ≥ 10 Desired level of genome coverage DNA purity and fragmentation most promoters very high high FFPE high molecular weight samples and pure Figure 2. An example of how the algorithm can be applied. Figure 2. An example of how the algorithm can be applied. 2. Profiling Whole Genome Methylation 2. Profiling Whole Genome Methylation Some broad examples of situations in which global genome methylation changes include [10]: Some(1) broadevents examplesthat impact ofthe situationsDNA (de)methylation in which machinery global genome [11,12]; (2) methylation the treatment changes of cells with include [10]: compounds, such as furan or azacytidine [13]; (3) cellular changes in brain tissue induced by (1) events that impact the DNA (de)methylation machinery [11,12]; (2) the treatment of cells with learning [14] and epigenetic changes that contribute to tumorigenesis [15,16]. Section 1 will describe compounds,six methods such by as which furan such or differences azacytidine can be [13 revealed]; (3) (represented cellular changes by Circle in 1 in brain Figure tissue 1). induced by learning [14] and epigenetic changes that contribute to tumorigenesis [15,16]. Section1 will describe six methods2.1. HPLC by which-UV such differences can be revealed (represented by Circle 1 in Figure1). The technique of HPLC-UV (high performance liquid chromatography-ultraviolet), developed 2.1. HPLC-UVby Kuo and colleagues in 1980 [17], is still considered to be the current “gold standard” assay for quantifying the amount of deoxycytidine (dC) and methylated cytosines (5 mC) present in a Thehydrolysed technique DNA of HPLC-UV sample. However, (high the performance utility of this liquidmethod chromatography-ultraviolet),is significantly limited by the need for developed by Kuo andspecialized colleagues laboratory in 1980 equipment [17], and is still the requirement considered of to relatively be the large current quantities “gold (3– standard”10 μg) of the assay for quantifyingDNA the sample amount to be of analysed deoxycytidine. Briefly, (dC)the DNA and must methylated be hydrolysed cytosines into its (5 constituent mC) present nucleoside in a hydrolysed DNA sample.bases, the However, 5 mC and dC the bases utility separated of this chromatographically method is significantly and, then, the limited fractions by measured. the need Then, for the specialized 5 mC/dC ratio can be calculated for each sample, and this can be compared between the experimental µ laboratoryand equipment control samples. and the requirement of relatively large quantities (3–10 g) of the DNA sample to be analysed. Briefly, the DNA must be hydrolysed into its constituent nucleoside bases, the 5 mC and dC bases2.2. LC separated-MS/MS chromatographically and, then, the fractions measured. Then, the 5 mC/dC ratio can be calculatedLiquid chromatography for each sample,coupled with and tandem this canmass be spectrometry compared (LC between-MS/MS) is the an experimentalalternative and control samples.high-sensitivity approach to HPLC-UV, which requires much smaller quantities of the hydrolysed DNA sample. In the case of mammalian DNA, of which ~2%–5% of all cytosine residues are 2.2.