Methylation-specific PCR - Reproducibility (Aug/19/2010 )

Hi

I’m new in the field of methylation-specific PCR (MS-PCR).
I’m using MS-PCR to investigate methylation at imprinted loci.
I use EZ DNA methylation-Gold Kit (Zymo Research) to bisulfite convert my DNA (a 2.5 hour reaction as recommended by the company). I have obtained the primer sequences from another laboratory and they appear to work well in my hands as well. However I’m wondering how reproducible my MS-PCR results are.

I’m interested in calculating the unmethylated/methylated ratio of my DNA samples. How big differences in this ratio should I expect/tolerate if in run 2 identical PCR reactions with DNA from the same sample (and from the same bisulfate reaction)?

Some of my results are quite reproducible but sometimes I see differences in the ratio of 0.8-1. Is that way too much? And if so, what can be the explanation?

Thanks in advance

XCI

I don't think it is reliable to calculate a methylation ratio using MSPCR as you suggest,

I would expect the variation between reactions (PCR) and the efficiency of the methyl specific and non-methyl specific are different and could mask the real changes or states you are trying to measure.

A direct sequencing approach or even pyrosequencing would be a better way to go because both alleles are measured in the same reaction

nick