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Optimizing methodologies for PCR-based DNA methylation analysis
 
Hernán G. Hernández1,2, 3, M. Yat Tse4, Stephen C. Pang4, Humberto Arboleda2, and Diego A. Forero1
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MSP is a simple method that requires resources commonly available in a molecular genetics laboratory and, once standardized, is effective for detecting methylated or unmethylated alleles without quantification. Processing up to 24 samples for both primer sets using conventional MSP requires about 4 h. Commercially available PCR master mixes for MSP (EpiTect MSP Kit, Qiagen, Hilden, Germany) are available; however, only conventional PCR reagents, including Hot-Start Taq polymerase, are required for the setup of MSP (43). While MSP assay kits are not commercially available, the MethPrimerDB database is available for help in selecting MSP primers (44).

Several real-time PCR adaptations of MSP also have been developed, including MethylQuant, a common option based on the measurement of increased fluorescence from SYBR Green I (45), and a real-time MSP approach combining conventional qPCR measurements with an additional melting step to detect amplicons associated with incomplete DNA conversion (46) or to distinguish the methylation status of individual alleles by comparison with standards of known allelic methylation status for an SNP located in the amplicon region (47).

Primer design considerations: As described by Herman et al., both methylated and unmethylated MSP primer sets should be designed to anneal to the same CpG containing region. MSP primers should include abundant CpG sequences at the primer binding sites to provide maximal discrimination between the methylated and unmethylated alleles. For the same reason, these CpGs should be as close as possible to the 3′ region of the primer (11) (Figure 2C). Additionally, a high number of thymines derived from non-CpG cytosines should be included to ensure specificity for converted DNA. MSP primer design is facilitated by the software listed in Table 3.

Data analysis: An amplification product of the correct molecular weight on an electrophoresis gel can be interpreted as methylated or unmethylated, depending on the specific primers used (11). The presence of amplification products using both sets of primers indicates a sample with both methylated and unmethylated DNA in the ROI (Figure 2D). However, a band from a reaction with methylated-specific primers might be a false positive. To avoid misinterpretation, inclusion of unmethylated DNA, non-converted DNA, and no-template negative controls is required (46,48). Likewise, the absence of an amplicon could be due to issues with the PCR reaction and must be controlled for as well (49).

The primary limitation of this technique is that it is qualitative (11). In general, well-standardized MSP assays provide information restricted to three possible outcomes: (i) presence of a methylated allele, (ii) presence of an unmethylated allele, or (iii) presence of both alleles. In assays intended to test MSP sensitivity, several ratios of the methylated and unmethylated DNA were used as templates. The results showed no clear correspondence between band intensity and dilution ratio, with many cases exhibiting very similar bands even for disparate levels of DNA methylation (50). On the other hand, several MSP assays demonstrated high sensitivity, detecting methylation percentages (MP) as low as 0.1% (50 pg of methylated DNA out of 50 ng of total DNA) or 1% (0.1 ng of methylated DNA out of 10 ng of total DNA) 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 to avoid amplification of non-converted DNA using MSP primers (11,52). Kristensen et al. identified false positive MSP results due to incomplete bisulfite-conversion, which is particularly problematic if only four or fewer non-CpG cytosines are included in the primer binding region (52). This issue has been associated with the apparent low reproducibility of numerous MSP assays (46,53). On the other hand, even after PCR amplification, MSP results can be validated by means of pyrosequencing to confirm the full conversion of every non-CpG cytosine (49). In MSP, PCR for methylated-specific or unmethylated-specific primer sets can frequently be standardized with non-identical PCR conditions (for example, different annealing temperatures) (11), possibly through inherent differences in sequence composition between primer sets. Therefore, identical PCR conditions for both MSP primer sets are not required for accuracy (11).

Real time PCR-based methods

MethyLight

Dual TaqMan labeled probes were developed for genotyping studies several years ago (54). Eads et al. subsequently introduced the use of TaqMan technology to determine DNA methylation status in specific genomic regions, a technique that was named MethyLight (55). Peter Laird's group defined four types of MethyLight reactions, depending on which oligonucleotides are designed to discriminate the methylation status: (i) only the primers, (ii) only the TaqMan probe, (iii) both primers and probe, or (iv) none (in cases where a control reaction is required to discriminate the converted DNA) (55) (Figure 3A). Using these reactions, several variations of MethyLight have been proposed to address different biological questions, such as the amount of methylated versus unmethylated alleles (56) or methylation status at the CpG dinucleotide level (which would be very expensive) (55). The most commonly used MethyLight methodology uses two primers and a TaqMan probe designed to bind the methylated allele specifically and requires a reference gene for normalization (55) (Figure 3A). It is important to note that MethyLight, depending on the method subtype, can assess the methylation status of all CpG sites covered by the TaqMan probes.

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