TITLE: Quantitative analysis of methylation and hydroxymethylation using oXBS-qMSP
AUTHORS: Hector Hernandez-Vargas, Chloe Goldsmith

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">There are several tools for investigating the methylation status of genomic DNA; many of these are expensive, do not allow for quantification or are unable to distinguish intermediary methylation metabolites such as 5-hydroxymethylation. Since significant changes in methylation status can be very small, the development of highly sensitive techniques is important for validating high throughput base resolution techniques. To this end, we developed a quantitative methyl specific PCR method based on oxidative bisulfite converted DNA. By designing primers specific for methylated and unmethylated DNA we are able to quantify the "real" methylation (5mC) and the hydroxymethylation (5hmC) at specific loci. This technique is important for understanding the methylation status of a region quickly and also as a tool for validating methyl-seq data. </div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/d352c3313980b1fdb419f493e4a783f5ed23e2a999af561044a623722e992d77/bn2744m6.jpg" /></div></div>

[STEPS]
?. [Preparing Control DNA]
Fully methylated, fully hydroxymethylated and fully unmethylated stanandards (synthetic DNA controls)In addition to the fully methylated and unmethylated controls, it is also important to have a control for the oXBS conversion. For this, it is possible to use synthetic DNA controls in order to efficiently quantify 5hmC. This DNA is unique since each cytosine in the respective samples is either methylated, unmethylated or hydroxymethylated, and not just in the context of CpG's. The primers for methylated and unmethylated were designed manually and sequences are provided in the table in the methods section, which, should be ordered along with the sequences designed for target regions.
NOTE: These synthetic DNA standards come fully methylated, unmethylated or hydroxymethylated, however, they still need to be BS and oXBS converted (step 5) the same as all DNA samples as well as the fully methylated and unmethylated controls.
?. [Preparing Control DNA]
Fully unmethylated DNA control Several controls are necessary to ensure your experiments are working efficiently. Whole genome amplification is sufficient to synthesize unmethylated DNA controls. We recommend the Qiagen REPLI-g whole genome amplification kit. PCR amplification should be performed according to manufacturer’s instructions.
?. [Quantitative Methyl Specific PCR]
Preperation of standard curve for % Methylation etermine the specificity of the primers to amplify methylated DNA in an abundance of unmethylated and vice versa. Converted DNA can be prepared according to the following volumes for 1 qMSP reaction:  in unmeth0  ABC1% MethylationFM (µL)FU (µL)210010033037490.99.152.70.279.7360.80.089.92
ABC1% MethylationFM (µL)FU (µL)210010033037490.99.152.70.279.7360.80.089.92
NOTE: These values are for 1 reaction and should be multiplied by the number of technical replicates and sets of primers required. Alternatively, serial dilutions can be prepared by diluting the fully methylated in unmethylated by a third (1/3) for each dilution.
?. [oXBS conversion]
Oxidative bisulfite conversion of DNA samplesThe following link is to the Nugen oXBS protocol for conversion of DNA: https://www.nugen.com/sites/default/files/M01481_v3_User_Guide%3A_TrueMethyl_oxBS_module_5911.pdfIn this protocol, each sample of DNA is split into two so that, in parallel, two reactions are conducted; regular bisulfite conversion (BS) as well as oxidative bisulfite conversion (oXBS). For more information see the guidelines and graphical abstract in this protocol and follow the link above.  -  of each sample, control or standard should be carried through the Nugen oXBS protocol according to manufacturers instructions. For controls and standards, we recommend taking   through the oXBS and BS protocols to ensure you have enough DNA for optimization purposes. The samples should be finally made up to   after BS or oXBS conversion. - can be used for each qMSP reaction depending on starting concentration of DNA.
100 ng
1000 ng
1000 ng
200 µl
2 µl
8 µl
Once DNA has been oXBS converted and BS converted it can be stored at -20C until further analysis.
?. [Primer Design for region of interrest ]
Primer designFASTA records for the target regions should be identified and can be taken from UCSC genome browser. Primers for methylated and unmethylated DNA (MSP primers) can be designed using available online tools including methprimer.
NOTE: Primer design has a huge impact on the specificity of the reaction. It is important to know your target region well and follow the rules for primer design closely to ensure primer stability and the avoidance of secondary structures. It is also possible to design primers and probes for taqman assays to further increase specificity, however, the assay we are outlining here is designed as a fast and inexpensive tool. Therefore, we have optimized based on SYBR green and basic primers with sufficient efficacy (as indicated by our control experiments (figure 1).
?. [Quantitative Methyl Specific PCR]
Calculation of Beta ValuesThe obtained Ct values can be used to calculate the % methylation at each loci according to the following analysis for the calculation of Beta Values: 2^- (CT values obtained by qPCR for Methylated primers) = M2^- (CT values obtained by qPCR for Unmethylated primers) = UBeta Values = M/(M+U) 5mC = Beta Value for oXBS5hmC = Beta Value for BS – Beta Value for oXBS Note: Beta Values Should be a value between 0 and 1. Negative Beta Values can be considered as 0.
Expected result for synthetic DNA after oXBS-qMSP analysis. Graphical representation of Beta Values obtained from synthetic DNA standards where all cytosine’s in the sequence are either unmethylated, methylated or hydroxyl-methylated. Bisulfite converted DNA represents the combination of methylated and hydroxymethylated cytosines (red bar), while oxidative bisulfite represents the true amount of methylated cytosine’s (pink bar), hence the hydroxyl-methylated cytosine’s can be deduced by difference between BS and oXBS (white bar).
?. [Preparing Control DNA]
Fully methylated DNA control
DNA can be methylated to make a fully methylated control by using the enzyme SAM (S-adenosylmethionine) (ZYMO cat# E2010). Ideally the unmethylated DNA synthesized in the last step should be used for this reaction.
?. [Preparing Control DNA]
The following link is to the detailed protocol for WGA needed to prepare a fully unmethylated sample. Starting material 1-10ng of DNA. The steps are outlined in the sub-sections to follow, however, reading the original protocol before-hand is advised. https://www.qiagen.com/fr/service-and-support/learning-hub/technologies-and-research-topics/wga/replig-principal-procedure/#REPLIg-protocols
?. [Preparing Control DNA]
Standard Reaction Setup: The setup below outlines a typical CpG methylase reaction in a 20 µl final reaction volume. The reaction volumes can be adjusted accordingly depending on experimental requirements. Prepare reagents according to the following:   AB12Reagent:µL3&nbsp; 10X CpG Reaction Buffer24&nbsp; 20X SAM
(S-adenosylmethionine), 12 mM15&nbsp; DNA at 100-250 ng/µl (1000ng maximum)46&nbsp; CpG Methylase (4 units/µl)17&nbsp; Water12
AB12Reagent:µL3&nbsp; 10X CpG Reaction Buffer24&nbsp; 20X SAM
(S-adenosylmethionine), 12 mM15&nbsp; DNA at 100-250 ng/µl (1000ng maximum)46&nbsp; CpG Methylase (4 units/µl)17&nbsp; Water12
?. [Preparing Control DNA]
Incubate at
[for 4 hours]
?. [Preparing Control DNA]
Add an additional  20X SAM (S-adenosylmethionine), 12 mM
1 µl
?. [Preparing Control DNA]
Incubate at
[for 4 hours]
Fully methylated DNA can be stored at  until further analysis.
?. [Quantitative Methyl Specific PCR]
qMSP is performed according to the following for 1 reaction:   AB1Reagent:µL2iTaq Universal SYBR Green Supermix103&nbsp; Forward Primer (10µM)1 (final concentration 100-500nM: varies depending on optimisation)4&nbsp; Reverse Primer (10µM)1 (final concentration 100-500nM: varies depending on optimisation)5&nbsp; Template2-86Nuclease free watermake up to final volume7Final volume20
AB1Reagent:µL2iTaq Universal SYBR Green Supermix103&nbsp; Forward Primer (10µM)1 (final concentration 100-500nM: varies depending on optimisation)4&nbsp; Reverse Primer (10µM)1 (final concentration 100-500nM: varies depending on optimisation)5&nbsp; Template2-86Nuclease free watermake up to final volume7Final volume20
NOTE: For each sample you will need to prepare 6x the reactions. Since 1 sample (in parallel) is BS converted and oXBS converted to calculate 5hmC by difference and triplicate technical replicates are the minimum suggested to ensure reproducibility of reactions.
?. [Quantitative Methyl Specific PCR]
Amplify DNA according to the following qPCR protocol:   ABCD12StageTemperature (°C)Time (min)31951542950.1553600.3goto 2 x 40 times6455-95, 0.5°C incrementsMelt Curve75END
ABCD12StageTemperature (°C)Time (min)31951542950.1553600.3goto 2 x 40 times6455-95, 0.5°C incrementsMelt Curve75END
?. [Quantitative Methyl Specific PCR]
EXPECTED RESULT FOR FULLY METHYLATED STANDARD CURVEPlot the values obtained for each concentration of DNA (Ct) against the log(starting concentration). This should be conducted for each set of primers to determine efficacy.
NOTE: Plotted values from standard curve should yield a straight line with a high R2 and a slope of approximately 3.2 for both the methylated and unmethylated primers. If this is not the case, new primers should be designed or optimized further for DNA and primer concentrations and annealing temperatures.
?. [Preparing Control DNA]
Prepare sufficient Buffer D1 and N1 for the total number of reactions seperately   ABC1ComponentBuffer D1Buffer N12Reconstituted Buffer DLB9 µL-3Stop solution-12 µL4Nuclease-Free water32 µL68 µL5Total Volume41 µL80 µL
ABC1ComponentBuffer D1Buffer N12Reconstituted Buffer DLB9 µL-3Stop solution-12 µL4Nuclease-Free water32 µL68 µL5Total Volume41 µL80 µL
?. [Preparing Control DNA]
Place   of template into microcentrifuge tube with   of Buffer D1 (prepared in step 2.2)
5 µl
5 µl
?. [Preparing Control DNA]
Incubate at room temperature for
?. [Preparing Control DNA]
Add   of the buffer N1 (prepared in step 2.2) and mix by vortexing
10 µl
?. [Preparing Control DNA]
Prepare master mix (MM)  on ice by adding  of REPLI-g Mini DNA Polymerase to of REPLI-g Mini reaction Buffer
1 µl
29 µl
?. [Preparing Control DNA]
Add MM from step 2.6   to denatured DNA Total volume now
30 µl
20 µl
50 µl
?. [Preparing Control DNA]
Incubate at  for And then inactivate polymerase by heating to   for
30 °C
65 °C
Amplified DNA can be stored at    until further analysis.
?. [Primer Design for region of interrest ]
Once the sequence for target region of interest is obtained, go to the methprimer website to design primers for MSP and select "Design PCR primers" (Pink)
?. [Primer Design for region of interrest ]
Paste the FASTA record into the available space and select "Pick MSP primers"
Here you can enter specifics for the primers including desired melting temps, product size ect. Default settings for MSP should be sufficient for most cases. An essential consideration for MSP primers is they must include at least 1 CpG site in the sequence in order to detect if there is methylation or not. More information can be found in the help section for MethPrimer.
?. [Primer Design for region of interrest ]
Selecting the Primer sequencesMeth Primer will suggest primer sequences for both methylated and unmethylated DNA. It is important to order both the methylated and unmethylated primers since two PCRs will be performed per sample. One for the methylated and another for the unmethylated.
Once you have selected the ideal primer sequences for your experiment, the oligos can be ordered through your preferred platform such as Invitrogen and optimised for melting temp and concentration as with regular primers for qPCR.