optimizing methodologies for pcr-based dna methylation analysis.pdf

7/25/2019 Optimizing methodologies for PCR-based DNA methylation analysis.pdf

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DNA methylation is the most extensivelystudied mechanism or epigenetic generegulation (1). Recent studies have shownthat DNA methylation plays an importantrole in a number o physiological processesas well as common diseases such as cancerand neurodegenerative disorders (2,3). Inmammals, DNA methylation occurs at theC-5 position o cytosine in CpG dinucle-otide sequences (Figure 1) (1), which aremainly concentrated in regions known asCpG islands. Methylation in CpG islandswithin gene promoters usually leads to genesilencing. More recently, DNA methylationin regions located up to 2 kb rom knownCpG islands (called CpG island shores) hasalso shown a strong correlation with geneexpression (4).

At present, the vast array o platormsavailable to study DNA methylation presenta challenge or scientists who wish to enterthis ield (5). Among the methods orstudying DNA methylation in candidateregions, PCR-based approaches haveseveral advantages (6). Here we providea practical overview o experimentaldesign and analysis or the most commonPCR-based DNA methylation techniques:bisulfite sequencing PCR (BSP), methyl-ation specific PCR (MSP), MethyLight,and methylation-sensitive high resolution

melting (MS-HRM). Tese techniques donot need expensive specialized equipmentand could be implemented in a typicalmolecular genetics laboratory.

Bisulfite conversionTe first step in almost all protocols orstudying DNA methylation is bisulfiteconversion o the DNA sequence o interest.Bisulite conversion occurs through anumber o chemical reactions (e.g., sulo-nation, deamination, and desulonation) onthe DNA that transorm non-methylatedcytosines into uracils. Methylated cytosinesremain unconverted (Figure 1). ClassicalDNA conversion protocols are time-

consuming, ofen requiring more than16 h to complete (7), and require multipletube changing steps that increase the risk ocontamination and human error. Classicalprotocols also risk losing more than 75%o the starting DNA (8,9) during purifi-cation and through single-strand breaksthat occur during long incubation steps(7,9).

Commercially available bisuliteconversion kits improve recovery o theconverted DNA by using shorter incubationsteps and alternative purification proce-dures (9). Tese kits also acilitate efficient

implementation o the conversion reaction,thereby improving downstream results

with PCR-based techniques. Tus, kitsare highly recommended, especially orthose unamiliar with this field o study.Tere are many considerations or selectinga kit, including cost, yield, efficiency, andtime. A comparison o the main eatures oavailable DNA conversion and methylationcontrol kits is included in ables 1 and 2.

Controls for DNA bisulfite conversionEvaluation o the quality o convertedDNA is recommended when beginninga DNA methylation study; this step isespecially important or quantitativePCR-based methods such as MethyLightand MS-HRM. Since bisulfite-treatmentcan result in DNA ragmentation, thusreducing the number o molecules availableor PCR amplification, it is best to test thebisulfite-converted DNA with primer setsthat ampliy a range o dierently sizedproducts. From these products, the idea lamplicon length or downstream analysiscan be determined (10), providing inor-mation that will aid in primer design.

Incomplete bisulite conversionwill adversely a ect the rel iabi lity andaccuracy o DNA methylation measure-ments by PCR-based methods (11,12).

Optimizing methodologies for PCR-based DNA

methylation analysisHernn G. Hernndez1,2,3, M. Yat se4, Stephen C. Pang4, Humberto Arboleda2, and Diego A. Forero11La borator y of NeuroPsy chiatric Geneti cs , Bi omedic al Sc ie nces Research Group, School ofMedicine, Unive rsidad Antonio Nar io, Bo got , Colombia , 2Neurosc ie nces Research Group,School of Medicine, Universidad Nacional de Colombia, Bogot, Colombia, 3Biomedical SciencesDoctoral Program, School of Medicine , Universidad Nacional de Col ombia, Bogot , Col ombia,4Department of Biomedical and Molecular Sciences, Queens Universit y, Kingston, ON, Canada.

BioTechniq ues55:181-197 (October 2013) d oi 10.2144/000114087

Keywords: epigenomics; DNA methylation; MS-HRM; MethyLight; 5-methylcytosine; polymerase chain reaction; reerence standards

Comprehensive analysis o DNA methylation patterns is critical or understanding the molecular basis o many hu-

man diseases. While hundreds o PCR-based DNA methylation studies are published every year, the selection andimplementation o appropriate methods or these studies can be challenging or molecular genetics researchers notyet amiliar with methylation analysis. Here we review the most commonly used PCR-based DNA methylationanalysis techniques: bisulfite sequencing PCR (BSP), methylation specific PCR (MSP), MethyLight, and methyl-ation-sensitive high resolution melting (MS-HRM). We provide critical analysis o the strengths and weaknesseso each approach as well as a series o guidelines to assist in selecting and implementing an appropriate method.

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Tereore, it is necessary to evaluate theefficiency o conversion using commer-cially available primer sets to ampliy theconverted DNA (e.g., DAPK1 Catalog

#D50142, Zymo Research, Orange, CA)(able 1). he resulting DNA productcan be sequenced to veriy the efficiencyo conversion or all non-CpG cytosines.Alternatively, converted DNA may beamplified with primers designed or thenon-converted DNA sequence. In this case,the absence o a PCR amplicon suggests acomplete conversion reaction.

Converted DNA must also be quantifiedprior to downstream PCR applications. Teamount o DNA may be determined byspectrophotometric measurements usingthe NanoDrop 2000 spectrophotometer

(Termo Scientific, Waltham, MA) (13)with settings or single-stranded DNA,or agarose gel electrophoresis and classicalUV spectrometric analyses (5). Other morespecific methods, such as qPCR (includingMethyLight control assays) or PicoGreenmay be more reliable and better suited ormeasuring limited amounts o DNA (14).

Designing primers for PCR-basedDNA methylation analysisDesigning primers against a region ointerest (ROI) is the most critical step inobtaining adequate DNA methylation

results using PCR-based methods. Severalsofware platorms such as Methyl PrimerExpress (Applied Biosystems, Foster City,CA), MethPrimer (15), BiSearch (16),MethMaker (17), and MSPprimer (18)have been developed or this purpose. Allo these programs allow users to customizeprimer length, amplicon length, and m(melting temperature) differences, as wellas enable searches or CpG islands in theinput sequence, and identiy possiblestable primer-dimer or hairpin structuresthat should be avoided. Te advantagesand disadvantages o each program are

compared in able 3. Primers should notbind to regions containing common SNPs(19), which can be identified easily using theUCSC Genome Browser (http://genome.

ucsc.edu).Because bisulfite treatment decreases

DNA sequence complexity, primers havean increased tendency to bind multipletarget sequences in converted DNA (18).Tereore, in silico evaluation o primerspecificity is a key step during primer designor bisulite-converted DNA methods.BiSearch sofware is unique in terms oits ability to find the number o potentialmatches, including partial matches, oreach individual primer in the bisulfite-converted methylated or unmethylatedgenome and to perorm in silico PCR on the

bisulfite-converted human genome usingany primer pair (16). In our laboratory, wehave observed greater success using primerswith less than 3000 matches.

At present, the sofware available orprimer design does not account or PCRbias (2022). When aced with bias, it isimportant to use additional tools to reviewthe ROI sequence, highlight the CpG andnon-CpG cytosines, and design adequateprimers. BioWord is a ree Microsof Wordplugin that allows manipulation, editing,and processing o DNA sequences and hasproven useul or working with sequences

prone to PCR bias (23). Another availableoption is a shareware version o the licensedsofware FastPCR, which includes a tool orin silico bisulfite conversion o non-CpGcytosines (24).

PCR-based techniquesBisulfite sequencing PCRBisulfite sequencing PCR (BSP) was theirst technique described or analyzingDNA methylation status using PCR (25).Te technique consists o PCR ampliyinga bisulfite-converted DNA ROI, ollowed

CGNNCNNNCNNCGCNCG

Methylated DNA

Non-converted sequencePost bisulfite treatment

sequences

CH3

CH3 CH3

CGNNCNNNCNNCGNNCG

Non Methylated DNA

CGNNUNNNUNNCGUNCG

UGNNUNNNUNNUGUNUG

Figure 1. Outline of bisulfite conversion. Non-methylated cytosines are transformed to thymines.N represents a nucleotide unchanged by bisulfite treatment. A light blue U represents a uracil

derived from bisulfite conversion of non CpG cytosines. A red U represents a uracil derived frombisulfite conversion of non-methylated cytosines in a CpG dinucleotide. Methylated cytosines in aCpG dinucleotide are not modified by the bisulfite conversion reaction. The CpG dinucleotides and

the UpG dinucleotides derived from the bisulfite conversion reaction are in bold and underlined.

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by Sanger sequencing o the product eitherdirectly or afer cloning into a suitable vector.Direct-BSP:By comparing sequencingresults with the respective reerencegenomic DNA sequences, direct sequencingo PCR products provides inormation onthe average methylation status or each CpGdinucleotide. Direct-BSP is the shortestorm o BSP, but holds several technicalchallenges inherent in sequencing, such aspoor signal quality and artiacts in cytosinesignals that may affect electropherogramanalysis; it also has a low sensitivity (26).Because o these diiculties, BSP withcloning is more common.Cloning-based BSP: In cloning-basedBSP, PCR products are cloned into a vectorand transormed into competent E. coli

cells. Afer expansion and purification othe plasmids, the PCR product inserts aresequenced. Te CpG methylation statusor each CpG dinucleotide in the ROI isdetermined by sequencing each expandedclone (27,28). Te resulting averages arereerred to as DNA-methylation haplo-types. Cloning-based BSP requires at leastsix sequencing reactions to obtain a sensi-tivity higher than direct BSP (29), makingthis an expensive and labor-intensiveoption that is especially cumbersome orpopulation-based studies.Digital (single-molecule) BSP:AnotherBSP option or producing DNA-methyl-ation haplotypes is digital-BSP (22,30).Tis method requires serial dilution o aDNA template to optimize conditions or

PCR amplification o a single convertedDNA molecule per reaction tube (via thePoisson distribution), thus avoiding bothPCR bias and cloning. Digital-BSP isconsidered the gold standard or detectingthe methylation status o specific loci (22).However, this method is inefficient because87% o the reactions cannot be analyzed,and 3% are control reactions; thus, useulinormation is only obtained rom theremaining 10% (22). An alternativeapproach is to use MS-HRM to select theclones or sequencing (31).Primer design considerations:Methyl-ation independent PCR (MIP) primersshould be designed to allow the ampli-ication o bisulite-converted DNAregardless o methylation status. Primers

Table 1. Comparison of commercially available kits for bisulfite conversion (single column format)

Provider

(References)DNA Input DNA Output (1)

Time

(2)Additional notes Link (3)

Reactions/

Cost (4)

Citations (5)

search Words

ZymoDNA Methylation Gold Kit (D5005

& D5006)

500 pg 2 g(Optimal 200

500 ng)

10 ml>99%>75%

4Modified DNA can

be stored at20C for to1 month

goo.gl/zAwu150 rxns (2.7)200 rxns (2.3)

1.380Cells-to-CpG-Bisulfite

Conversion

QiagenEpiTect Bisulfite Kit (59104)

1 ng 2 g20 ml>99%

NA6

Protocol for FFPESamples

goo.gl /nNTeu 48 rxns (4.4)1.020

EpiTect- Bisulfite-Kit

Qiagen

ZymoDNA Methylation Direct Kit

(D5020 & D5021)

50 pg- 2 g(Optimal 200 -

500ng)

10 ml>99.5%>80%

5Protocol for FFPE

Samplesgoo.gl/JJnDI

50 rxns (3.6)200 rxns (2.5)

95Zymo DNA Methylation-

direct-kit

ZymoEZ DNA Methylation-Lightning

Kit (D5030 & D5031)

100 pg - 2 g(Optimal 200 -

500ng)

10 ml>99.5%>80%

3For long-term use

store at -70Cgoo.gl/zAwu1

50 rxns (3.6)200 rxns (2.5)

0Methylation Lightning-kit

Zymo

EpigentekBisulFlash DNA Modification Kit

(P-1026050)

0.2 ng 1g (Optimal

200500 ng)

>90%>99.9%

2,5Protocol for DNA

input 0.2 ng/50 cellsgoo.gl/TU79j 50 rxns (2.2)

7Epigentek BisulFlash DNA

Modification Kit

Chemicon (Millipore)CpGenome Turbo BisulfiteModification Kit (S7847)

500 pg- 1 mg(Optimal 1 ng

-1mg)

25 ml>99.9%

NA3

Modified DNA canbe stored at 20C

for to 2 monthsgoo.gl /xMNDz 50 rxns (3.3)

21CpGenome Bisulfite

Modification-Kit-Millipore-fast

Chemicon (Millipore)CpGenome Fast DNA

Modification Kit (S7824)

1 ng - 1 mg(500 pg of DNA

can be used)

3545LNANA

24Modified DNA canbe stored at 20C

for to 2 monthsgoo.gl/3sern

T25rxns (8.0)17

CpGenome Bisulfite fast

Modification-Kit-Millipore

EpigentekMethylamp Modification Kit

(P-10011 & P-10012)

1 ng-1 g.(Optimal

50200 ng)

818 L99.5%

NA

4Modified DNA canbe stored at 20C

for to 2 months

goo.gl/7ElOC40 rxns (2.7)80 rxns (2.5)

67Methylamp DNA-

Modification-Kit -Coupled-DNAEpigentek

Methylamp DNA Isolation &Modification Kit (P-100240)

1 ng-1 g.(Optimal

50200 ng)

818 L>99.9%>90%

3Protocol for adhesivecells, plasma, serum,

and body fluidsgoo.gl/IoOvy 40 rxns (5.0)

1Methylamp-Coupled

Epigentek

New England BiolabsEpiMark Bisulfite Conversion Kit

(E3318S)

50 ng2 mg 40 ml 4Modified DNA canbe stored at 20Cfor up to 2 months

goo.gl/kKRLk 48 rxns (2.8)8

EpiMark Bisulfite-

Conversion-Kit)

Sigma-AldrichImprint Bisulfite

DNA Modification Kit (MOD50)

10 ng to 1 mg(Optimal 10 ng

- 1 mg)

820 L>99%

NA3

50 pg / 20 cellsprotocol

goo.gl/IUhOI50 rxns (4.3)

46Imprint-DNA-Modification-

Kit Sigma

InvitrogenCells-to-CpG Bisulfite Conversion

Kit (4445555)

50 pg-5g(Optimal 100

ng - 1 g)

10 L-4099.5%

NA7

Protocol for blood,10 - 105cells (optimal

5000 -105cells)goo.gl/upJ1A

50 rxns (5.0)1

Cells-to-CpG-Bisulfite-

Conversion-kit

Human Genetic SignaturesMethylEasy Xceed

Human Genetic Signatures

50pg -5 mg(12 L -100 L)

50 l>99,9>90%

2,5Protocol for inputequivalent to 8human cells

goo.gl/4JuMI40 rxns (6.5) 52

MethylEasy Xceed

Diagenode

MagBisulfite Magnetic bisulfiteconversion kit (AF-1060024)

>1 ng

(Optimal 100ng-1g)

50 l

>99%NA

4,5 Magnetic beadpurification

goo.gl/QZ2JN 24 rxns (13.6) 0MagBisulfite + Diagenode

Clontech.EpiXplore Methyl Detection Kit

(631968)

50 pg5 mg12100 ml

NANA

2,5


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should also not bind regions containingCpG dinucleotides (Figure 2A) (25) andshould flank a sequence o converted DNAcontaining as many thymines originatingrom the conversion o non-CpG cytosinesas possible (25). Guidelines or designingBSP primers were initially published byClark et a l. in 1994 (25).

Recently, several more techniques havebeen developed using primers based onthe same principles (32). One variant othe direct-BSP method uses two roundso nested PCR with primers designed bystandard methods or the first round andprimers with a GC-rich tag at the 5 end orthe second round. Tis primer modificationis intended to reduce non-specific amplifi-cation during direct-BSP and compensateor the requent artiacts seen in direct-BSPresults (33,34).Bias in BSP:Several studies using MIPprimers have shown bias toward unmeth-

ylated or methylated alleles (8,20), likely dueto sequence differences between methylatedand unmethylated al leles (34). For example,Warnecke et al. ound a 33-old amplifi-cation bias toward the unmethylated allelewhen assaying a region o the RB1 genepromoter (20). In some cases, adjust ingMgCl

2concentrations or redesigning the

primers to bind the opposite DNA strandmay be sufficient to resolve this bias (20,34).

Shen et al. ound that, in some instances,adjusting the annealing temperaturemay correct this bias (35). Wojdacz et al.developed a new approach to primer designthat allows the use o annealing temper-ature changes to adjust or amplificationbias (36,37). Tese new guidelines or biascompensation are described in detail in the

MS-HRM section o this article.Another potential source o error occurs

in cloning-based BSP methods. Cloningbiases may skew the reliability o resultsgenerated rom BSP assays (22,34). Tere isevidence that amplicons without cytosinesmay be more difficult to clone efficiently (8).Although direct-BSP has low sensitivity, itprovides more accurate detection o differ-ences as low as 20% in methylation statusin a single CpG (29).Data analysis:uantification o methyl-ation levels is determined by comparingthe relative peak heights o cytosine and

thymine (or adenine and guanine in caseso complementary strand sequences) (25)in each CpG position in the electrophe-rogram (Figure 2B). A qualitative analysiso bisulite sequencing results can beperormed i a clear single peak is presentor each CpG cytosine position. In that case,a thymine peak would be interpreted as anon-methylated CpG, and a cytosine peakwould represent a methylated CpG. Analysis

o raw sequence data rom direct-BSP isofen difficult, but correction algorithmsaid data interpretation. he EpigeneticSequencing Methylation Sofware (ESME)program includes an algorithm to analyzedirect-BSP sequencing results and providesa qual ity control filter (able 4) (29).

ESME may also be used to analyze

cloning-based BSP electropherograms (29).In cloning-BSP or digital-BSP, satisactorysequencing results belonging to the samesample should be averaged to determine thelevel o methylation or each CpG position.Tis task is acilitated by BiQ-Analyzer andBISMA (38,39) (able 4).BSP selection: Different BSP method-ologies are optimal or different methyl-ation studies, depending on the particularconditions o a study and other param-eters, including cost, research question, andavailable samples. For example, the studyinitially validating digital MethyLight

is a case where digital-BSP was the bestchoice, since it allowed accurate validationo another single-molecule technique andthe use o automated PCR-processing ora large number o sequencing reactions(30). In many other cases, cloning-BSP ispreerred because it is the only option ordetermining DNA-methylation haplotypesin general laboratories (34). Direct-BSPwas selected to assess DNA methylation

Table 2. Comparison of commercially available kits for DNA methylation controls

Provider (Reference) Amount / Cost (1) Features Notes Link (2) Citations (3)

Millipore

S8001(S8001M & S8001U)

5g / 20L$ 212

Methylated DNA

HCT116 DKO cells DNA methylated by

M.SssI DNMT (EC 2.1.1.37)>95% of CpGs methylated

Non-Methylated DNA

HCT116 DKO cells DNMT1 (-/-)DNMT3b (-/-)


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CGNNNNTTNCNNCNCNNCGCNCGTN

CGNNNNTTNCNNCNCNNCGCNCGTN

CGNNNNTTNUNNUNUNNCGUNCGTN

UGNNNNTTNUNNUNUNNUGTNUGTN

Non-methylated DNA

Forward Primer: NNNNTTNTNNTNTNN

Reverse Primer : NNANANNANAANNNN *

CH3 CH3 CH3

--------------------------------

--------------------------------

------------------------------

-------------------------------

Post-bisulfite sequence binding regionPre-bisulfite sequence binding region

Methylated DNA

Non-methylated DNA

Methylated DNA

T

T

C

T T

CpG1 CpG2 CpG1 CpG2

C C C C

Cloning-based BSP Direct BSP

C

C

Y (60%)

Y (30%)

TC

CpG1 CpG2

T

C

T Y (20%)

Y (50%)

Y (80%)

Y (50%)

C: Methylated (100%)

T: Non-methylated (0%)

NNCGNNCNCNNCGCNCG

Methylated DNA

NNCGNNCNCNNCGNNCG

Non Methylated DNA

NNCGNNUNUNNCGUNCG

NNUGNNUNUNNUGUNUG

MSP Primer design

NNCGNNTNTNNCGTNCG

NNTGNNTNTNNTGTNTG

Post-bisulfite sequence ampliconPre-bisulfite DNA sequence Forward primer design

Post-bisulfite sequence ampliconPre-bisulfite DNA sequence Forward primer design

CH3 CH3 CH3 CH3 CH3 CH3

Methylation

level

100%

Methylation

level

0%

Methylation

level

0,1%-99,9%

ElectrophoresisPossible results

M U M U M U

MSP assay

C D

Figure 2. Primer design and results for bisulfite specific PCR (BSP) and methylation specific PCR (MSP). (A) BSP primer. The dashed line indicates the se-quence of the primer binding region in this example. Note that CpGs are avoided in the design of this kind of primers. *For simplicity, we use the same bind-

ing sequence of the forward primer to illustrate a hypothetical reverse primer design. (B) Simplified electropherogram schema of possible results from BSPfor two CpG cytosines. Left panel: Cloning-based BSP. Possible sequencing results for a single clone. The possibilities are reduced to methylated (remainsC on the sequencing data) or unmethylated (T replaces C in comparison with non-treated sequence). Right panel: Direct BSP C denotes methylated status(100%), T denotes non-methylated (0%) and Y (C + T) denotes different methylation percentages. (C) Chart of an example of MSP primer design over a bind-ing site specific for methylated DNA (top) and non-methylated DNA (bottom). The untreated sequence (left) is modified depending on its DNA methylationstatus (middle). This sequence is used to design the forward primer by substituting the Us with Ts (D) Diagram of the possible results of an MSP assay on anelectrophoresis gel. N: non-affected nucleotides in bisulfite treatment. The Us and Ts in red represent the uracils derived from bisulfite conversion of CpG cyto-sines and the corresponding thymines in the primer sequence. The CpG dinucleotide and the corresponding UpG or TpG sequences are in bold and underlined.

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in BDNF to complement initial resultsrom MSP screening experiments (40).Beore deciding between direct-BSP andcloning-BSP or a particular application,we recommend testing and compa ringpreviously validated primers and strategies(26,27,33).

Methylation Specific PCRMethylation speciic PCR (MSP), irstdescribed by Herman et al. in 1996,determines the methylation status o anROI through selective ampliication omethylated and unmethylated alleles. Tetwo-tube approach employs two primer sets:one binding specifically to the methylatedsequence and another binding to theunmethylated sequence (11,41) (Figure 2C).A two-round variant o MSP, reerred to asnested-MSP (N-MSP), has been describedand can be used in special cases (42).

MSP is a simple method that requires

resources commonly available in a moleculargenetics laboratory and, once standardized,is eective or detecting methylated orunmethylated alleles without quantification.Processing up to 24 samples or both primersets using conventional MSP requires about4 h. Commercially available PCR mastermixes or MSP (Epiect MSP Kit, Qiagen,Hilden, Germany) are available; however,only conventional PCR reagents, includingHot-Start aq polymerase, are required orthe setup o MSP (43). While MSP assaykits are not commercially available, theMethPrimerDB database is available or

help in selecting MSP primers (44).Several real-time PCR adaptations oMSP also have been developed, includingMethyluant, a common option based onthe measurement o increased fluorescencerom SYBR Green I (45), and a real-timeMSP approach combining conventionalqPCR measurements with an additionalmelting step to detect amplicons associatedwith incomplete DNA conversion (46)or to distinguish the methylation statuso individual alleles by comparison withstandards o known allelic methylation

status or an SNP located in the ampliconregion (47).Primer design considerations: Asdescribed by Herman et al., both methylatedand unmethylated MSP primer sets shouldbe designed to anneal to the same CpGcontaining region. MSP primers shouldinclude abundant CpG sequences at the

primer binding sites to provide maximaldiscrimination between the methylated andunmethylated alleles. For the same reason,these CpGs should be as close as possible tothe 3 region o the primer (11) (Figure 2C).Additionally, a high number o thyminesderived rom non-CpG cytosines should beincluded to ensure specificity or convertedDNA. MSP primer design is acilitated bythe sofware listed in able 3.Data analysis: An amplification product othe correct molecular weight on an electro-phoresis gel can be interpreted as methylatedor unmethylated, depending on the specific

primers used (11). Te presence o amplifi-cation products using both sets o primersindicates a sample with both methylatedand unmethylated DNA in the ROI (Figure2D). However, a band rom a reactionwith methylated-specific primers mightbe a alse positive. o avoid misinterpre-tation, inclusion o unmethylated DNA,non-converted DNA, and no-templatenegative controls is required (46,48).Likewise, the absence o an amplicon couldbe due to issues with the PCR reaction andmust be controlled or as well (49).

Te primary limitation o this technique

is that it is qualitative (11). In general,wel l-standard ized MSP assays provideinormation restricted to three possibleoutcomes: (i) presence o a methylated allele,(ii) presence o an unmethylated allele,or (iii) presence o both alleles. In assaysintended to test MSP sensitivity, severalratios o the methylated and unmethylatedDNA were used as templates. Te resultsshowed no clear correspondence betweenband intensity and dilution ratio, withmany cases exhibiting very similar bandseven or disparate levels o DNA methyl-

ation (50). On the other hand, severalMSP assays demonstrated high sensitivity,detecting methylation percentages (MP)as low as 0.1% (50 pg o methylated DNAout o 50 ng o total DNA) or 1% (0.1 ngo methylated DNA out o 10 ng o totalDNA) in different studies (11,43,50).Possible challenges:Low quality DNA is

associated with a decrease in reproducibility(51). As mentioned above, it is critical toavoid amplification o non-converted DNAusing MSP primers (11,52). Kristensen etal. identified alse positive MSP results dueto incomplete bisulfite-conversion, whichis particularly problematic i only our orewer non-CpG cytosines are included inthe primer binding region (52). Tis issuehas been associated with the apparent lowreproducibility o numerous MSP assays(46,53). On the other hand, even afer PCRamplification, MSP results can be validatedby means o pyrosequencing to confirm

the ull conversion o every non-CpGcytosine (49). In MSP, PCR or methylated-specific or unmethylated-specific primersets can requently be standardized withnon-identical PCR conditions (or example,dierent annealing temperatures) (11),possibly throug h inherent differences insequence composition between primersets. Tereore, identical PCR conditionsor both MSP primer sets are not requiredor accuracy (11).

Real time PCR-based methodsMethyLight

Dual aqMan labeled probes were developedor genotyping studies several years ago(54). Eads et al. subsequently introducedthe use o aqMan technology to determineDNA methylation status in specific genomicregions, a technique that was named Methy-Light (55). Peter Lairds group defined ourtypes o MethyLight reactions, dependingon which oligonucleotides are designed todiscriminate the methylation status: (i)only the primers, (ii) only the aqManprobe, (iii) both primers and probe, or (iv)

Table 3. Freely available software for primer design for methylation analysis

ToolMSP /BSP

design

Design for bias

compensation

Primer thermody-

namic evaluation

Additional

advantageAdditional disadvantage

In silico PCR

on bisulfte

treated DNA

Link

Bi-Search yes no yesAble to test the

primer specicity

It does not inform the cause of primer

rejection in in-silico PCR

yesbisearch.enzim.hu

MethPrimeryes no

no Simple and

exible

No automatic function to search for primers

reverse complementary to the input sequenceno

www.urogene.org/methprimer/

Methyl Primer Express

(Applied Biosystems)yes no

no Many customable

featuresNo very intuitive no www.expbiosystems.com

Perl primer yes no yesCustomable CpG

island denitionBugs on the copy-paste function no perlprimer.sourceforge.net/

MSPPrimeryes no yes

Primer design with

higher specicity

Nested MSP

design included

User registration is necessary No www.mspprimer.org


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www.Bioechniques.comVol. | No. |

none (in cases where a control reaction is

required to discriminate the convertedDNA) (55) (Figure 3A). Using thesereactions, several variations o Methy-Light have been proposed to addressdierent biological questions, such as theamount o methylated versus unmeth-ylated al leles (56) or methylation statusat the CpG dinucleotide level (whichwould be very expensive) (55). he mostcommonly used MethyLight method-ology uses two primers and a aqManprobe designed to bind the methylatedallele speciical ly and requires a reerencegene or norma lization (55) (Figure 3A).

It is important to note that MethyLight,depending on the method subtype, canassess the methylation status o all CpGsites covered by the aqMan probes.

he quantitative aspect o Methy-Light has been explored since itsinception. Analyses using dierentratios o methylated to unmethylatedDNA have been employed to veriy thelinearity o this quantitative assay, witha high linear correlation ound betweenthe dilution ratios and the MethyLightMP measurements (55,57). MethyLighthas shown higher levels o accuracy

and lower rates o alse negatives whencompared with previously describedtechniques (45,55). For this reason,MethyLight is requently used to validateother techniques or DNA methylationstudies (36). However, unlike MS-HRM(36), the most commonly used Methy-Light technique cannot detect hetero-geneous methylation in a sa mple becausethe MethyLight primers and probes aredesigned to measure a speciic methyl-ation pattern (ully methylated).Primers and probe design consider-ations:In a MethyLight assay, it is necessary

to normalize each qPCR reaction using sets

o primers and probes that bind a convertedDNA region independent o its methylationstatus (Figure 3A, top). Tereore, eachMethyLight assay should include both anROI amplification reaction and an MIPreaction or the control region. Currently,commercially available Beacon Designersofware (Primer Biosof, Palo Alto, CA)is the method o choice or MethyLightprimer design. Tis program is able to designMethyLight assays, but in practice it isrestricted to CpG islands, possibly becauseCpG density in the island shores and insome promoters lacking islands is low.

Data analysis:Depending on the Methy-Light design, relative fluorescence units(RFUs) should be used to calculate themethylation percentage. For the commonMethyLight design described in this section,the broadly accepted determination ormulaor DNA methylation percentage is shownin Equation 1 (55).

In order to evaluate the methylationstatus o an ROI using MethyLight, it isbest to select a previously validated primer/probe set or that ROI i a similar researchquestion is to be addressed. Houshdaran etal. (58) have perormed ~300 MethyLight

assays and have made the primer and probesequences available.Example of MethyLight selection:Methy-Light is the technique o choice whena study requires accurate quantitativeassessment o DNA methylation. It hasbeen used in a number o cancer associatedDNA methylation studies, including thedevelopment o an assay to measure thepresence o methylated alleles in three genes

associated with colorectal cancer (59). Here,

the assay was ocused on clinical applica-tions o cancer detection. It should be notedthat the cost o using aqMan probes can behigher than other real-time PCR methodsthat utilize cheaper intercalating dyes (46).his is o particular importance i thesample size o the proposed study is large,or i a significant number o ROIs is to beassessed.

Methylation-sensitive highresolution meltingIn the DNA double helix, a cytosine anda guanine o complementary strands are

linked by a triple hydrogen bond whilea thymine and an adenine are joined bya double hydrogen bond (36). Tereore,base composition can directly influencethe thermodynamic behavior o DNA in amelting analysis. m is defined as the temper-ature at which the PCR product dissociatesinto two single strands and a sharp drop influorescence o a DNA intercalating dye isobserved (37). Tis basic principle can beused to discriminate between methylatedand unmethylated alleles ollowing bisulfiteconversion. Distinction between allelesis achieved through m analysis o the

MIP-PCR products in the ROI, in whichthe methylated allele usually has a higher mthan the unmethylated allele (60) (Figure3D).

Initially, Worm et al. (61) described anin-tube melting protocol or analyzing DNAmethylation prior to the development ohigh resolution melting (HRM) technology.Afer technical improvements in meltingassays, Wojdacz et al. developed a DNA

Table 4. Multi-purpose sofware or DNA methylation test design a nd analysis o results.

Tool Method Function Web link

BioWord

For all PCR-based

DNA methylation

techniques

Editing, replacing and assistance for primer design http://sourceforge.net/projects/bioword/

FastPCRIn-silico analysis of several designed primers, bisulte conversion of non-

CpG cytosineswww.biocenter.helsinki./bi/programs/fastpcr.htm

MethGraph Automatic genomic representation of the designed primers http://mellre.ugent.be/methgraph/

MethBlastIn-silico evaluation of oligonucleotide sequence similarities to bisulphite

modied genome sequenceshttp://medgen.ugent.be/methBLAST/methBLAST_cs.php

MethMakerGenerate possible assays for the supported experimental methods in the

ROI http://methmarker.mpi-inf.mpg.de/Beacon

Designer

software

MethyLight Primer/probes design and primer evaluation www.premierbiosoft.com/molecular_beacons/index.html

ESME Direct BSP Analysis of results www.epigenome.org/index.php?page = download

BiQ Analyzer Cloning-based BSP Analysis of results http://biq-analyzer.bioinf.mpi-inf.mpg.de/download.php

BISMA Cloning-based BSP Analysis of results http://biq-analyzer.bioinf.mpi-inf.mpg.de/download.php

Poland

MS-HRM Tm Calculation for PCR amplicon

www.biophys.uni-duesseldorf.de/local/POLAND//poland.html

MELT http://web.mit.edu/osp/www/melt.html

OligoCalc www.basic.northwestern.edu/biotools/oligocalc.html

The software displayed in this table are freely available online with two exceptions: Shareware with full functionality available only for a limited time. Licensed software, demo version with limited functionality.

100standard)methylated(FullygenegnormalizinamountinputMIP

standard)methylated(FullySTATUSMethylatedgeneROITarget/

(Sample)g)normalizinamountinput(DNAMIP

(Sample)statusmethylatedofsignalROIx

Equation 1.


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MethyLight Assay

Methylated CpG

Non-Methylated CpG

Fluorescent dye extreme of MethyLight probe

Quencher dye extreme of MethyLight probe

NGCAAG CNNANNANGCNGCANGCAN

NCGTTCGNNCNNCNCGNCGCNCGTN

From Methylated DNA

From Non-Methylated DNA

NCGTTCGNNTNNTNCGNCGTNCGTN

Pre-bisulfite amplicon sequence

NTGTTTGNNTNNTNTGNTGTNTGTN

Example of amplicon between primers

G G G G G

Post-bisulfite amplicon sequence

0 % Methylated DNA

Methylated Standard

-d(RFU)/dT

Temperature (Celsius degree)80

500

1500

2500

Rel

ative

SignalDifference

Temperature (Celsius degree)

76 78 80

0

20

40 60%

50%

40%

30%

20%

10%

90%

80%

70%

100%

0%

10

30

50

70

60

NGCAAG CNNANNANGCNGCANGCANA A A A A

Evaluated

sample

76 78

NGCAAG CNNANNANGCNGCANGCANG G G G GG G

MS-HRM Assay

A B

C D

Fluorescent dye extreme of MethyLight probe (reactions for

non-methylated DNA)

Fluorescent dye extreme of MethyLight probe (DNA amount

normalization-reaction)

MIP primers on

100% Methylated DNA

MIP primers on the

evaluated sample

ROI primers on

100% Methylated DNA

ROI primers on

the evaluated sample

Fluorescence

Cycle

www.Bioechniques.comVol. | No. |

methylation assay implementing HRMtechnology: the methylation-sensitivehigh-resolution melting (MS-HRM)techniques (36). Te MS-HRM method-ology consists o real-time PCR usingbisulfite-converted DNA (regardless o themethylation status) and melting analysiso PCR products (HRM) to discriminate

the ROI methylation status reflected in thethermodynamic behavior o the MS-HRMamplicon.

he MS-HRM method enablesassessment o the percentage o themethylated allele present or a particularsample in an ROI. Tis is possible throughcomparison with melting standard curvescreated by dierent dilution ratios omethylated and unmethylated DNAcontrols (37). Since the technique analyzesthe melting properties o the final PCRproducts, MS-HRM not only evaluatesully methylated alleles in proportion to

ully unmethylated ones, but is also able todetect heterogeneously methylated samples(62).PCR bias:As or other MIP based amplifi-cations, potential PCR bias or MS-HRMwas evaluated during development o thetechnique (21). MS-HRM showed a strongamplification bias toward unmethylated

sequences when the classic recommenda-tions or primer design stated by Clark etal. were ollowed (25). In contrast, usingthe recommendations o Wojdacz et al.or primer design (21), variations o theannealing temperature in the PCR cyclingstep allowed or control o PCR bias (21).Monitoring o real-time PCR amplificationestablishes an additional quality control stepor MS-HRM experiments (13). Similar todigital-BSP, digital MS-HRM is also useulor reducing PCR bias (62). Consideringthe possibility o PCR bias, it is importantto highlight that quantitative methyl-

ation analysis with MS-HRM is based onthe assumption that methylation levels oCpG sites between the primers is the sameas methylation levels o CpG sites coveredby the primers.Primer design considerations:MS-HRMprimer desig n ollows the same generalprinciples o classic MIP design as previ-

ously detailed by Clark et al . (25). However,in order to compensate or PCR bias, thereare new recommendations or MS-HRMprimer design that advise inclusion o oneor two CpG annealing sites (located as aras possible rom the 3 end o the primers toavoid methylation specific amplification)(Figure 3C) (60). Currently, there are noprograms or MS-HRM primer design thatincorporate the new recommendations tocompensate or PCR bias. Finally, severalprograms such as OligoCalc, Poland, andMEL (able 4) can predict the meltingcurves o the PCR products.

Figure 3. Real time PCR assays: MethyLightand MS-HRM. (A) Schematic lollipop graphof MethyLight subtypes. The most usedapproach consists of primers and probes

designed for converted methylated DNA se-quences and uses MIP primers to normal-ize the DNA input (top). Another choice is to

design a set of primers and probes specific

for methylated DNA and another set specificfor non-methylated DNA (middle). In these

cases, the sum of both signals is used tonormalize the individual signals. Similar toclassic MSP, one primer set is specific for

methylated DNA and the other set for un-methylated DNA (bottom). One probe, de-signed to bind DNA independent from its

methylation state, is used with both primersets. The normalization procedure is similarto the one for the middle section. (B) Ampli-

fication curve of the most used MethyLightsubtype (top in A). The RFU value determi-nation for 100% methylated control DNA al-

lows calculation of the percentage of meth-ylated molecules in the evaluated sample.The MIP signals allow an adequate control

of DNA input amount for both the evaluatedsample and 100% Methylated DNA. (C) Sim-plified outline of an MS-HRM amplicon for

the analysis of methylation status. The triplehydrogen bond of G and C is represented.(D) On top: schematic graph of the negative

first derivative of the melting-curve. DNAthat is 0% methylated has a lower meltingtemperature peak in comparison to 100%

methylated DNA. Non-converted DNA hasthe highest number of triple hydrogen bondsand therefore presents the highest melting

peak. On bottom: schematic plot of the dif-ferences for the normalized signal of thestandard curves. The plot also presents a

curve for an illustrative sample that is locat-

ed between the 10% and 20% standard di-lution curves. (In this type of melting curve,

0% of methylation is used as the clusterof reference.) Conventions as in Figure 2.

A B

C D


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Data Analysis:Wojdacz et al. (36,37)prop osed a met hod or estimat ingmethylation levels by comparing themelting patterns o standard templates

with known proportions o methylatedand unmethylated DNA controls to themelting patterns ound in a sample. Tesemiquantitative estimate is based onsimilarities in HRM patterns without amathematical approach or calculatingthe DNA methylation percentage. Morerecently, se et al. (2011) implementedan MS-HRM approach to quantiy themethylation status o each sample withhigh reproducibility. Peak-height and areaunder-the-curve rom the normalized,temperature-shited dierence curveswere used to generate linear standard

curves (13) (Figure 3D). uantitative datawere obtained by interpolation o the firstderivative o the normalized melt curves,generated by the linear regression analysiso the standard curve (13). When hetero-geneous DNA methylation patternsare present in a sample, HRM analysiswill identiy such heterogeneity by thecomplex shape o the melting curves;however, in such cases quantitative HRMmeasurement is not possible (62). Tepresence o SNPs in the amplicon regioncould generate additional variations inthe melting profiles (37).

Examples of MS-HRM selection:MSP-based assays only evaluate DNAmethylation or CpG sites present in theprimer binding reg ion (usual ly 80 bp), regardless o the methylationstatus o CpGs within the primer bindingsite (36). Tereore, MS-HRM providesthe ability to evaluate a larger genomicregion when compared with MSP-relatedtechniques (Figure 4) (11). MS-HRM isa good choice or quantitative determi-nation o DNA methylation levels, whensequence level detai l is not required (63). Agood example o the usage o MS-HRM isa colorectal cancer study where the authorsdistinguished different stages o the disease

and their correlations with the quantity oDNA methylation (64).

Proper controls for PCR-basedDNA methylation analysisIn addition to the controls used in conven-tional PCR assays, other steps shouldbe taken to veriy the accuracy o DNAmethylation data generated in PCR-basedassays. Unconverted genomic DNA is anessential control that should be included inall optimization processes or PCR-basedDNA methylation assays; it providesinormation on the ampliication o

Table 5. Comparison of PCR-based DNA methylation techniques

Basics Advantages Disadvantages

Direct BSP MIP primers to amplify the region of interest (ROI)

(for the population of cells that conform the

sample) and sequencing

Information of methylation at CpG resolution level

One sequencing reaction for sample

No information at single molecule level

Possible PCR bias (no detectable)

Considerable noise in sequencing results

Cloning-based BSP MIP primers to amplify the ROI (for the population

of cells that conform the sample), cloning and

sequencing reaction for each clone

Information of methylation at CpG resolution level

Information at single molecule level

Reduced noise in the sequences reaction

PCR bias

Cloning procedure (time and money consuming)

PCR bias (no easily detectable)

Possible cloning bias (no detectable)

At least 5 sequencing reactions for sample

Single molecule

(digital) direct BSP

MIP primers to amplify the ROI using serial dilutions of

template to obtain single molecule PCR products

Sequencing the positive amplications to obtain a

number of single molecule sequences

Quantitative information of methylation at

CpG resolution level

Information at single molecule level

Reduced noise in the sequences reaction

No PCR bias

No cloning bias

Serial elusion step to determine the most effective

input for each PCR reaction

At least 25 sequencing reactions for sample for

acceptable sensitivity

5 fold increase in sequencing reactions for one

sequencing result

Classical MSP MSP primers, PCRs and electrophoresis

CpG site at or near the 3 primer allow

amplication of only methylated or

unmethylated DNA

Stringent annealing temperatures to avoid

amplication of unconverted DNA

High sensitivity

May be very cost-effective in settings where

the objective is detection of any degree of

methylation at primer binding region

No sequencing reaction required

Low specicity

Not quantitative

False-positive results (no easily detectable)

Two separate tube reactions for assay

No information of methylation at CpG resolution

level neither at single molecule level

Real Time Based

MSP

MSP primers

CpG site at or near the 3primer allowamplication of only methylated DNA or

unmethylated DNA

Stringent annealing temperatures to avoid

amplication of unconverted DNA

Feasibility of a semiquantitative approach

Very high sensitivity

Information of methylation at primer binding region

No sequencing reaction

No quantitative (semiquantitative approach)

and low specicity

Two primer sets, each set used in one separated tube

reaction (possibility of different amplication

efciencies)

No information of methylation at CpG resolution level

No information at single molecule level, unless using

digital approach

MS-HRM MIP / primer set for compensation of PCR bias

One step PCR and HRM

High sensitivity, a quantitative approach

One primer set in a closed tube assay

Information of methylation at regional level

Immediate detection of false-positive amplication

and PCR bias


level

No information at single molecule level, unless

using digital MS-HRM

MethyLight Different kinds of Methylight approaches.

Refer to text for particular advantages and

disadvantages of each kind of MethyLight

Very high sensitivity

Closed tube assay

Quantitative approach

No information at single molecule level unless

using digital MethyLight MethyLight


level, unless use of CpG specic probe


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non-converted DNA with primers that arespecific or the converted DNA. For specificassays, amplification rom bisulfite-treatedDNA should show a clear difference romany possible result using non-converted

DNA.In one o the pioneering MSP studies,Herman et al. verified primer specificity orthe bisulfite modified p16 sequence usinguntreated DNA in reactions with eithermethylated-speciic or unmethylated-specific primers (11). As expected, no ampli-fication was ound when non-convertedDNA was used as a template. Nonetheless,several reports o MSP standardization didnot include or report this kind o control(65,66).

Similarly, the use o non-convertedDNA is also recommended when using the

MS-HRM technique during assay optimi-zation (37). Tis type o control is the easiestto include but, paradoxically, is the controalmost commonly omitted or not reported(63). It allows experimental verification othe specificity o the assay or convertedDNA. In most cases, there should be noamplification products; however, in someinstances products will be amplified thatcan be easily identified when compared withthe converted DNA (37).

Use o ully methylated and unmeth-ylated DNA is a critical experi mentalcontrol as well. It should be noted that

DNA considered to be ully unmethylatedcomes rom a variety o different sources.he practice o using DNA obtainedrom peripheral blood mononuclear cells(PBMC) as a ully unmethylated DNA

control is valid in cases where the samplesare indeed completely unmethylated atthe loci o interest. Several reports haveocused on detecting DNA methylationstatus in peripheral blood, showing biolog-ically important methylation levels ormultiple genes (52,67). For example, lowlevel methylation o many cancer-relevantgenes may be ound in the PBMCs romnormal individuals. Tereore, the indis-criminate use o DNA rom PBMCs as anegative control in sensitive assays or DNAmethylation detection may be particularlyproblematic (52).

Manuacturers o commerciallyavailable DNA controls have dierentstrategies or providing ully methylatedand unmethylated DNA. For example,ully non-methylated kits rom Zymo andMillipore use DNA rom cells that containgenetic knockouts o 2 key DNA methyl-transerases, thus reducing methylationlevels by more than 95% (68).

Fully methylated DNA can be obtainedrom M.SssI-methylated DNA rom, amongmany sources, double knockout cells orDNM1 and DNM3b (able 2). Anotheralternative is to use the product rom whole

genome amplification (WGA) with kitssuch as REPLI-g (Qiagen), which does notreproduce the DNA methylation patternand has a theoretical methylation level oless than 10-6. However, this amplificationapproach may carry the risk o reducedrepresentation o the loci o interest (69).Tereore, the use o identical amounts

o methylated and unmethylated controlsderived rom the same class o template(genomic DNA or WGA-products) couldguarantee that equivalent amounts oeffective templates are included.

DiscussionStudying DNA methylation or a candidateROI using PCR-based methods is a topico present and uture importance. Tereare many advantages o genome-wideplatorms; however, PCR-based techniquespermit detai led analysis o specific regions

o the genome, including CpG islandshores. In addition, the associated costs oimplementing and executing PCR-basedtechniques are lower, allowing the initialstudy o several candidate ROIs. PCR-basedapproaches also offer the advantage o alower burden o alse discoveries and theability to confirm a large number o ROIsidentified in genome-wide screening o aew samples (70).

DNA methylation analysis using pyrose-quencing is a quantitative approach thatdoes not require a cloning step, but presentsthe risk o PCR bias, similar to BSP. More

importantly, pyrosequencing instrumen-tation is not commonly available in a generallaboratory. For interested readers, compar-isons and discussions o pyrosequencingtechniques are available elsewhere (71).

Tis article highlights several consider-ations or PCR-based DNA methylationstudies. Te approaches reviewed here havedifferent advantages and disadvantages thatshould be evaluated beore starting anyDNA methylation study. Similarly, it is clearthat the different PCR-based techniquesdiscussed here have differences in CpGcoverage and possibility or quantitation

(Figure 4). A comparison o all PCR-basedDNA methylation techniques is presentedin able 5.

Several considerations concerningPCR-based methods or DNA methylationanalysis will be crucial or the consolidationo the field o molecular epigenetics. Currentneeds in this field include (i) detailed experi-mental comparisons o results obtained withdifferent PCR-based techniques (72), (ii)the availability o a large number o prede-signed PCR-based DNA methylationassays to acil itate broad use, (iii) the imple-mentation o minimum reporting guide-

BSP assay

MSP assay

MS-HRM assay

MethyLight assay

Non-evaluated CpGs

Evaluated CpGs

Fluorescent dye

Quencher dye

Figure 4. CpG coverage of PCR-based DNA methylation techniques.A simplified lollipop schemashows the CpG dinucleotides as circles. The methylation status is not represented. Grey circlesrepresent the non-evaluated CpGs in each corresponding technique. Red circles represent the

CpG dinucleotides evaluated in each method. In the MethyLight assay, the green oval representsthe fluorescent molecule and the black oval represents the quencher molecule.


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lines or manuscripts describing results oPCR-based analyses o DNA methylation,including details o experimental condi-tions such as controls, primer sequences,and programs used or primer design (73),(iv) the urther development o additionalPCR-based techniques that allow DNAmethylation measurements in a more

quantitative and reproducible way (5), and(v) the implementation o automatic andmultiplexed protocols or DNA methyl-ation using currently available techniquesto improve efficiency and reduce costs (59).For readers interested in genome-wide DNAmethylation analysis, we recommend twoavailable review articles (74,75). Finally, itis critical to keep in mind that the resultso PCR-based DNA methylation method-ologies are reliable only in an experimentalsetting with adequate methodologicalcontrols.

Ack nowledgmentshis work was supported by grants romColciencias (Contract # 401-2011),UAN-VCI, and UNAL-DIB. HGHis a recipient o a PhD ellowship romColciencias. he authors would like tothank the anonymous reviewers or theirimportant comments and suggestions.

Competing interests.Te authors declare no competing interests.

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Received 07 November 2012; accepted 10 September 2013.

Address correspondence to Diego A. Forero,

Laboratory of NeuroPsychiatric Genetics,Biomedical Sciences Research Group, School ofMedicine, Universidad Antonio Nario, Bogot,Colombia. E-mail: [email protected]

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