3 replies to this topic

[Emma Court]

Please could anyone suggest a method for extraction of RNA from resuscitated cryopreserved bone marrow cells. I have tried RNazol, Trizol, phenol-chloroform extraction and Nucleospin (Clontech) methods, which work fine using fresh cells but fail when viable, resuscitated cryopreserved cells are used.

[kimquinn]

Your expression "resuscitated cryopreserved cells' concerns me. Does this mean you are defrosting the frozen marrow prior to commencing RNA extraction? If so, you may be activating RNases and hence causing degradation and a very poor yield of RNA. You should commence homogenising / extracting your RNA from the deep frozen state directly into a chaotropic agent (guanidinium, Trizol etc.)

[AronD]

Emma,

In addition to kinmquinn's suggestion, you may want to consider how the samples were handled prior to freezing.  Blood and bone marrow have a very short window of RNA stability in the cell; within 24 hours you will have esentially no RNA.  If the samples were not frozen within 24 hours of collection, you may not be able to get any RNA from them no matter which method you use.

[Emma Court]

Many thanks for your suggestions. Just to clarify things, my aim is to revive program- frozen bone marrow cells (which have been shown to be viable for at least  48 hours post-thawing) and to stimulate them with certain cytokines, then extract RNA to assess cellular responses. I would greatly appreciate any further suggestions on how to achieve this, if indeed there is a solution. Samples were cryopreserved often within one hour and with great care, so I am unsure if lack of RNA is the problem. Other bone marrow samples have been revived, stimulated, then newly-synthesised protein extracted from them, so perhaps it is the inherent instability of RNA that is the problem (as you suggested). Either way, I would welcome further comments or suggestions.
Many thanks, Emma