Hi! I'm fairly new to using bisulfite conversions in the lab and have a general question about how I should be quantifying my bisulfite converted DNA. Typically our lab uses UV spectrophotometry (Nanodrop) to quantify nucleotides, however I've encountered people saying that this is not an effective way to measure concentrations post-bisulfite conversion as the product is neither typical DNA or RNA. I have currently been measuring concentrations on an RNA setting, but I was curious as to what other have done.
I also received a suggestion to measure concentrations of DNA pre-bisulfite conversion and normalize my concentrations at that point instead. The researcher told me that this should be sufficient, as I would be subjecting all of my samples to the same bisulfite conversion and thus concentrations should theoretically be the same. Has anyone found success in this method or are there any reasons that this would not work?
If you need any additional information to answer the question, we will be doing methylation-specific qPCR in order to compare methylation of specific genes across different experimental groups. Thank you!
You need not have to do quantification of the DNA. We use Ez DNA methylation kit from zymo research. We use 1.5ug of DNA for conversion and elute the converted DNA in 20ul. Typically we use 2 to 3 ul of converted DNA for pcr. If you want more details contact me at [email protected] or [email protected]
Hi Allison, I have done bisulfite conversion and methylation analysis on PCR products over a time course in plants ( Article The Rapid Methylation of T-DNAs Upon Agrobacterium Inoculati...
) . I used the EpiTect Fast DNA Bisulfite Kit (Qiagen) and would convert between 1250 and 1750 ng (keep this the same for each data set you want to compare). You have to assume complete conversion and minimal loss during the conversion process and as such you need to quantify the DNA before conversion. I have found this to work quite well as per the suggestion you received. If using the nanodrop post-conversion, I would use the single-stranded DNA setting.
Using either RNA or ssDNA settings on nanodrop might be the "best" option. However, neither of these would be a better option than the other since bisulfite converted DNA could be a mixture of ssDNA/RNA and/or dsDNA/RNA. Remember that bisulfite treatment will transform unmethylated Cs into Uracils (Us are transformed into Ts after PCR) and methylated Cs will remain the same. These changes will alter complementarity between DNA chains and some regions of these "transformed" DNA will have Us showing properties of DNA and RNA at the same time. Considering this, quantification of nucleic acids will vary depending on the settings used on the nanodrop, but the same setting must be used to quantify all the samples in the study. This will at least tell you which sample has more or less methylation within these conditions.
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