comparison of methods for bisulphite treatment - (Nov/20/2006 )
We have recently switched to using the Epitect bisulfite kit for bisulfite conversion of genomic DNA followed by Methylation specific PCR. Earlier we were using the Manual method i.e. the 16 hr treatment as proposed everywhere followed by DNA cleanup using Wizard system. We have always a positive and Negative control for all PCR's. We notice that while results are similar for our control/cell line DNA using both methods, now few very faint methylated bands that were visible by the 16 hr. treatment are not visible now using this kit which uses a PCR based method and completes the treatment in 6 hrs. In many cancers we only get very faint methylated bands for various reasons- are they important? That is my second question. The first of course is which method seems more trustworthy? We are using published primers for genes using methylation Specific PCR. My third and Final(!) question is regarding bisulfite sequencing-this is to validate our MSP results as for many patients and few genes it makes more sense to do screening using MSP Followed by bisulfite sequencing. Can we simply do PCR using bisulfite sequencing primers and directly sequence the products using the same primers after cleanup or do we have to clone in first in TOPO/OTHER vector?
Thanks you so much and look forward to your responses.
From memory I think the epitect kit involves a couple of denaturation/annealing steps to ensure the DNA to be treated is single-stranded. It is only when it is single stranded that bisulfite conversion takes place.
A faint M amplicon could suggest incomplete conversion with the homebrew method and the primers are misprining but; can also at the same time suggest a mixed population of cells in your initial sample.
If the MSP is optimised well it can be used for screening purposes on a large cohort of patients from which the interesting ones are taken to the next level in direct or, cloning then sequencing the amplicons.
I need to know which results to believe in more- epitect or manual. In manual 16 hr bisulfite protocol, all controls work fine so what can the faint methylated band in patients reveal?Of course in tumors we have normal cell population plus the differential pcr efficiency of methylated vs. unmethylated reactions. What does your experience say?Do we only count the thick meth bands as real?