I would like to freeze patient samples now for future epigenetic analysis. I would be interested in looking at changes in one or two cell populations, which is why I don’t want to take isolate total leukocyte DNA right now.
I was thinking about lysing whole blood with a RBC lysis solution, washing the cells then freezing then in a solution of RPMI, 5% FCS and 10% DMSO. The cells would then be cooled in a cryobox and transferred to liquid nitrogen until use.
I tried looking for articles describing epigenetic changes induced by DMSO, but most articles discussing it cultured the cells in DMSO for 4+ days.
Does anybody have any opinions on this or possibly know of any articles discussing epigenetic changes in frozen cells?
In my experience, DNA methylation is a quite stable mark, specially if you always keep your cells at low temperatures. I mean, if after thawing them you are always strict to keep them on ice, epigenetic machinery (nor cell metabolism in general) won't be really active.
I'd rather avoid the step of frosting and thawing, and perform the population separation ASAP, but I guess that if you are not doing it is for a reason (lack of the antibodies to do it, not decided which cell populations to isolate, etc..)
We have compared the DNA methylation levels of blood samples (CD4+ T and CD19+ B cells) isolated on the same day, to others isolated after leaving the blood 2 days at room temperature, and at least DNA methylation was quite robust (which didn't happened for miRNAs).
Hope this helps!
Best,
Lorenzo
How about this article?
Riesco MF, Robles V (2013) Cryopreservation Causes Genetic and Epigenetic Changes in Zebrafish Genital Ridges. PLoS ONE 8(6): e67614. doi:10.1371/journal.pone.0067614
It seems cryopreservation does affect some targets. If you have an idea of what cell type you want to look at, you could start with a pilot experiment (preserved vs. fresh) in a cell line to analyze a few candidate genes for epigenetic changes. Also, you can try other cryopreservation conditions (lower DMSO, propylene glycol, 90% serum, etc) if you're not satisfied with the results. Then of course you would need to validate on patient samples.
Let us know what you find!
Sylvia
In our lab, in order to study DNA methylation changes we store at -80°C the dried pellet of cels (in aliquots of about 1-2*106 cells). Cells may be PBMCs (from fycoll separation) or CD4+, CD8+ and CD19+, NK cells (from MACS separation starting from PBMCs). From these aliquots DNA was extraxcted and methylation analysed. By, Laura
Thanks for the article Sylvia. I'd seen it before, but was wondering if anybody has had experience in human cells.
Thanks for the info Laura. Have you compared freshly isolated to cryopreserved samples?
interesting question. We routinely extract DNA from frozen whole blood for epigenome-wide association studies but we haven't done a direct comparison of fresh versus frozen fro the same individual. What we have done is a comparison of fresh vs. guthrie card extracted DNA and using the illumina 450K platform saw an ICC of 0.99 between the two samples. But both samples were extracted within a week of sample collection so if time to extraction is a factor that won't have come in to play.
This paper addresses freeze-thaw issues to some extent:
Genomics. 2010 Feb;95(2):73-83. doi: 10.1016/j.ygeno.2009.12.001. Epub 2009 Dec 18.
EBV transformation and cell culturing destabilizes DNA methylation in human lymphoblastoid cell lines.
Grafodatskaya D, Choufani S, Ferreira JC, Butcher DT, Lou Y, Zhao C, Scherer SW, Weksberg R.
Source
Hospital for Sick Children Research Institute, Toronto, Ontario, Canada.
PMID: 20005943
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