2 replies to this topic

[Swongel]

Hi all,

I would appreciate your advice on the following if you could please:

I would like to compare DNA methylation levels of samples from saliva and blood that came from the same individuals. I will be using the EpiTect Bisulfite kit to convert these samples and subsequently perform a High Resolution Melt Analysis (HRM) using Rotor Gene.

The problem I have is the starting DNA concentration for the saliva DNA varies and is very very low (i.e. ranges from [2.82ng/ul] to [22.3ng/ul]); however, the blood DNA samples are all equilibrated at 100ng/ul.  Normally when I do this type of work all my samples are at the same concentration level i.e. [100ng/ul]. Do you suggest I run the experiment with varying concentration levels? A colleague suggested to select the minimum concentration I have as a threshold (in this case [2.82ng/ul]) and dilute all the remaining samples (even those currently at [100ng/ul] down to this concentration level in order to start with a uniform concentration level and continue with the bisulfite treatment and HRM analysis.This would mean having all samples at [2.82ng/ul] :-s!

I'm a bit conflicted as part of me thinks 1) yes, it's better to start with a uniform concentration level, specially if I want to compare the two groups (saliva vs blood) and the other part thinks 2) no, whatever level of DNA concentration I start with the amplification of the DNA depends on the amount of reagent I have in each tube. I assume once the product is finished the fluorescence level would reach its plateau.

Any advice?

Thank you in advance

[paulcross]

If the DNA concentration is too low, after bisulfite treatment , you won't get any band.

[vilperte]

Samples with low concentration after bisulfite conversion are very difficult to amplify.

I had the same sample with 40ng/ul and with 4ng/ul. I really tried to amplify the one with the lowest concentration, but it was impossible.

I would suggest you to try a new bisulfite conversion (if you still have samples for that). Have you tried the bisulfite conversion with RNA carrier? The kit manual says that you don't need to use it if you start with more than 100ng. I made a test using the same sample with and without carrier, starting with 200ng. With carrier I got 40ng/ul and without I got 4ng/ul.