There have been long discussions on RNAlater composition, how to make a home-made RNAlater etc. but I'm wondering - and I would love to hear especially from those scientists who actually tried to compare some of the RNA preservants and then they checked the quality of the isolated RNA - how do they compare? Especially, I'm curious which one is the best for storage of field samples - that is for storing the samples for RNA isolation at ambient temperature for up to several days?
To share my recent observation - I have always used TRIzol preserved samples but now I was handling home-made RNAlater samples which spend several days at ambient temperature and I was surprised how degraded it was (when analyzed on Agilent Bioanalyzer). I have to stress that my comparison of the two preservants is far from being perfect since other conditions could have also influenced the my RNA integrity: the tissues were not exactly the same (although quite similar in both cases - insect whole bodies) and I also used slightly modified procedure for the isolation from samples stored in RNAlater vs. TRIzol.
It is easy to find the manufacturers description of the preserving capacity of the preservants but I was not able to really find any kind of comparison of the two procedures and I would be really interested in your experience! Thanks for any hints and pieces of info which will be hopefully useful also for others.
RNAlater is tricky to use... I make my own but if it isn't used properly in the field, there is nothing I can do to get better RNA. (I haven't tried preserving in Trizol- it sounds like it would be great, just unpractical for long distance field collections for plants.)
To get the best yield using RNAlater, break up your tissue to allow for maximum diffusion of the RNAlater. The chitin of the insects' bodies will make it difficult for the salts to diffuse and halt metabolic processes. (The same goes for plants with waxy cuticles etc.) Also, try to make sure that there is 10x the volume of RNAlater in respect to your tissue.
Try to refrigerate your samples as soon as possible after collecting. (My colleagues share their field cooler with their samples.) When they get to their final destination, refrigerate them over night (4C) before draining the RNAlater and freezing the samples.
You may have to modify your RNA extraction method to account for all of the salt, but RNAlater can work! Good luck!
Ultimately, trizol will be much more superior as it contains the GuSCN (thiocyanate) which inhibits RNAses on contact and of course also contains phenol which will pretty much keep things stable for a long time. From what I can tell, the RNAlater does not have it and therefore will never be as good. However, given the conditions you are facing, you may want to consider spiking your RNAlater with some GuSCN as a chemical means to inhibit RNAses. I don't know how well GuSCN can breakthrough chitin (or what is the best concentration) Its a bit toxic (no more than Trizol) but may help improve your storage conditions.
Dear all,
thanks for your suggestions!
Nicolas - thanks. Just to let you know - I've tried adding 0.8M GuSCN (concentration mentioned in the TriZol patent) as you suggested but I didn't see any improvement in the quality of isolated total RNA - at least under the conditions I used to simulate the harsh sample storage conditions in the field (I believe it still might help under different conditions).
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