Dear All, I would like to know what is most appropriate protocol for storage of intestine biopsies in Liquid Nitrogen?
1. Take sample. 2. Put it in per-chilled cryovial. 3. Immediately place cryovial in LN2 container.
Is there any better/more optimized approach then please share. I have to first store these samples for around 3-4 months and then process.
I think what you're describing is called snap freezing. If you are at the bench or don't have N2 immediately available, you can rapidly freeze tissue by filling a small container no more than a 1/3 full of ethanol and equal parts dry ice. Dip your cryovials with tissue in the liquid and it will freeze immediately. Then you can keep on dry ice until you reach the LN2 container. The ethanol will evaporate from off the outside of the tube. In any event make sure cap on cryovials is sealed completely.
hope this helps
Thanks for reply. I am performing sample collection from different hospitals and my institute is in another city, so I don't have easy access to my lab. I only have LN2 available but no other preserving reagent with me. I have to use these tissues for microarray study. I have came across several protocols online but then there are variations which are confusing me. So, I would like to know whether above mentioned steps in my earlier post are correct for sampling? Secondly, I have observed that once frozen, tissue stick to wall or bottom of cryovial. In case when I have to perform RNA isolation steps, would that be better to fill these cryovials with TRIZOL and then homogenize? Its my first time to perform these steps so please guide.
I can't speak to working with human tissue where more care might be necessary. With that said, I'm not certain pre-chilled cryovials are necessary since the freezing step is extremely fast, but yes the protocol looks fine to me. If you're sub-sampling from the cryovial for RNA extraction--and intend to use the rest of the sample for tissue culture or flow cytometry--then obviously adding TRIZOL, which lyses cells, is not good. It would seem easier to transport tissues in LN2 and do RNA extraction back in the lab. In field research, people often preserve tissue in pure ethanol. It's not as ideal as LN2 though.
hope this helps
I agree with James about not needing to pre-chill the cryovials if using a LN2 or a dry ice/ethanol bath.
You might also want to look into Ambion's RNA-Later reagent, it would probably be better than ethanol as a chemical fixative in the field.
http://www.invitrogen.com/site/us/en/home/brands/Product-Brand/rnalater.html
I believe that cooling such intestine tissue samples gradually within outside container containing warm water(25 degrees Celsius) until it comes to 5 degrees. Then adding 5% pure glycerol to the media that is added to the tissue will enhance the preservability of the tissue in LN2 until use for various biological determinations.
Moustafa has a point. If you want to do histological/immunohistochemical studies at any point, it is important to preserve the tissue in a way that protects structure. Glycerol should help this.
If all you need is an expression profile, I would stick with the RNAlater for field work.
I'd have to look and see if RNAlater is compatible with histological studies; I've never used it for this purpose before.
Found this information on AMBION webpage about RNAlater. They are referring to a study which have evaluated whether RNAlater is compatible with histological studies and appears to be Yes,
http://www.invitrogen.com/site/us/en/home/brands/Product-Brand/rnalater.html
Wafa , if you use DMSO, just be careful to use it while cooled (at 5C), otherwise it will hirt your cells. Also, you must use at a lower level than Glycerol.
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