I am working on methylation analysis of DNA (from blood/PBMC's). The technique which I will carry out is COBRA which is done by bisulfite conversion, followed by PCR amplification. Then based on the presence or absence of RE site restriction enzyme they make the cut. PAGE is carried out after that and then via densitometric analysis we can calculate the % of methylation.
However, few articles mention electroblotting as the technique of their choice instead of PAGE. Can either of you help me and let me know if I can do PAGE and still get the % of methylation instead of blotting.
Dear Banerjee,
Greetings!
Quantification of methylation in percentage rely upon the method adopted for cobra. If you have carried out allele-specific method, thus you must go for blotting and if you used non-allele-specific method, so as alternative approaches such as direct sequencing, bis-sulfate pyrosequencing etc. can be used. Further, You can search few dye to produce visible bands after page analysis, photographed and can manually score for -nce/+nce for the corresponding bands.
Rest, a healthy discussion can be raised.
Regards,
Dheeman S.
Hello,
Thank you for your answer. However, you have left me a little confused over here. I am planning for nested PCR of bisulfite converted DNA and then RE digestion and running gel (PAGE). the dye employed will be EtBr.
And, I was hoping to deduce the % by densitometric analysis. Do you think it is a correct approach?
Dear! Hello again
In your experiment, densitometric analysis of the blots (or band after the gel staining with appropriate dye; If EtBr is used UV-transilluminator or Gel-Doc can be used further to visualize the band and photograph directly) can be carried out using Image Quant TL (IQTL) software (GE healthcare). The basic steps involved in this analysis are as follows:-
1. Blot images are imported into the software and the contrast is adjusted such that the bands are clearly visible on the blot image.
2. Area around each band is selected.
3. Background intensity is subtracted from the blot image.
4. Bands are then selected by drawing a tight boundary around them. Intensities of the selected bands is then displayed in an excel format which can be exported for carrying out further statistical analyses.
Hello,
Thank you so much! I will for sure check out the software. Is it freely available?
Also, as you are aware I am looking at RE digestion of the Bisulfite converted DNA. Would you like to suggest some apt RE for the same?
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