Bisulfite treatment

A commercial bisulfite treatment kit (EpiTect Bisulfite Kit) is available from... 

Bisulfite treatment kits have been developed and are commercially available (e.g., Qiagen bisulfite treatment kit). Kits work with the same efficacy and have helped streamline the procedure, which will help expedite the process because of the elimination of setup time. Beginners are encouraged to use a kit, especially when there is not enough DNA to calibrate the conditions for bisulfite treatment. 

Bisulfite Modification of Genomic DNA Using a Bisulfite Treatment Kit... 

Quantify your DNA and dilute 100-2000 ng (based on the availability or number of the required experiments) in 20 pL water for bisulfite treatment according the manufacturer s instructions (e.g., EpiTect Bisulfite). 

Furthermore, MSP analysis shonld not yield any prodncts nsing the same amount of unmodified genomic DNA as the template. The presence of products would indicate that the bisulfite treatment is incomplete, which more likely will generate a false-positive signal for methylation. This may also indicate that yonr primers are not specific for the modified DNA (see clues for designing primers for MSP, 9.3.4). 

To obtain accurate results in qMSP/QM-MSP analyses, the best conditions have to be worked out to achieve reliable standard curves during the test trials. This could be achieved with the use of unmethylated and methylated templates such as placental DNA and in vitro methylated DNA, respectively, and by performing bisulfite treatment as described. To find the best condition for each gene, purify the DNA, calculate the concentration and copy numbers, and dilute the DNA sequentially (e.g., 1,1/2,1/4,1/8, 1/16, 1/32, and 1/64) and perform real-time PCR with several dilutions of the primers (e.g., 25, 50, 75ng each in various combinations). For example, you should see the amplification plots as indicated in Fig. 9.4 with 50% (Fig. 9.4A) or 25% (Fig. 9.4B) sequential dilution. These test trials and any other quantitative PCR (qPCR)ZQM-MSP experiments need to be done in duplicate or triplicate to ensure that the required skills and instruments for equal pipeting are in place, or the impacts could be minimized by averaging the results of the triplicate experiments. Similar to MSP, for each qMSP or QM-MSP trials use placental and... 

All measurements should be made between 15 min (see step 4) and 1 hr after sample preparation and bisulfite treatment. Longer standing times tend to increase observed readings. 

The bisulfite treatment removes any aldehydic material present at this point. Since this ketone is quite inert to sodium bisulfite, the yield is not lowered by this procedure. 

Finally, NGS can also be applied to epigenome analysis, currently one of the main areas of cancer research. By one of three different approaches, immunoprecipitation of methyl-cytosine after DNA fragmentation, capture of CpG islands and associated sequences, or complete genome sequencing after bisulfite treatment, all of them are directed to envision what promoter regions may be modified by methylation-based silencing (121,122). 

All dichloromethane examined showed 2-14 ppm of formaldehyde contamination. Several clean up methods were applied to remove formaldehyde such as washing with sodium bisulfite, treatment with active charcoal of Porapak Q porous polymer without success. Trace levels of formaldehyde in solvents may be impossible to remove. Therefore, chloroform was used as the solvent for formaldehyde analysis in further experiments. The amount of contaminant obtained from a blank solvent was always subtracted from the values of actual results. Dichloromethane was, however, used for methyl glyoxal analysis. The extraction efficiency of chloroform and dichloromethane was almost identical. Dichloromethane was easier to use for a liquid-liquid continuous extraction than chloroform because of its lower boiling point. 

Sulfonic acids can be prepared by direct sulfonation (see above, Figure 38.1). Compounds containing conjugated double bonds have been solubilized by addition of sodium bisulfite. Treatment of menadione (vitamin K3) with sodium bisulfite leads to two addition compounds (Figure 38.11). Mild warming of the reactants for a short time predominantly affords adduct (a) which arises from attack of bisulfite ion at carbon 2. Heating at reflux for an extended period yields adduct (b) from addition of bisulfite ion to carbon in position 3 . ... 

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