kit or manual bisulphite conversion dilemma - (Feb/25/2007 )

Dear All,
what is the verdict regarding a more sensitive approach to bisulphite conversion?the old fashioned manual 16hr protocol of Frommer et al.,or a fast pcr based kit one like the epitect one?your answers are very needed to help us believe our results specially in our archived patient pool.
thanks
meths

Hi meth

As far as I know the kits and "old fashioned" basically work after the same principle, so there is not much difference in conversion efficiency.
This might vary strongly though form kit to kit, so if you do decide to buy one make sure you're getting a good one.

A kit is more expensive, but will assure you that you always have the same reagents. It is also ready to use when you get it.
This means less establishment work to do, and also less "test" samples you will need to establish the method.

The "old-fashioned" way will give you more steps where you could possibly make errors. In most cases will take longer to establish, but it is cheaper.
There are also two protocols pinned in this section called "The actual bisulfite treatment" and "Bisulfite DNA modification protocol".
They work well and some of your quesitons may already have been answered oin those threads, so check there first!

In your case I would decide depending on three things:

a.) the time available (no time: use kit; lots of time: establish method)
b.) the amount of "test-tissue" you have available. (no or litttle tissue for testing method: buy kit; lots of extra tissue available: establish method)
c.) how long the lab will use the technique in future (BS conversion only this one time: buy kit; BS conversion will be used often: establish method)


Concerning kits, we used the "EZ DNA Methylation Kit" from "Zymo", and it worked well.
I've heard of the Epitect one on this forum as well, so it probably works fine too.

I'm sure some other people here know more about the differences between the kits and can recommend one

Just read that Qiagen EpiTect is he way to go concerning kits, just fyi

I put my hand up for MEthyleasy, big fan of it.

does low amounts of starting DNA no problem at all.

I have heard from as little as 5 cells you can get DNA for conversion.

Nick

From 5 cells? Incredible! Did they isolate the DNA before? Or is there a way to use Methyl Easy with agarose beads (cells are embedded)? I'm trying with this, but Methyl Easy has a step to precipitate the DNA what I don't want to do with my beads - I just put them directly into the PCR reaction. Last time I used the untreated Methyl Easy control DNA, I treated it with the "old-fashioned" manual way and finally it worked - though I used 35 cycles for both round of nested PCR and still get a poor lane... but at least it was there. I think I'm going to move on this way. now I'm treating some of my genomic DNA, so cross fingers for me...