Snap freezing tissue for RNA isolation - (Dec/10/2007 )

Hi,

I am planning a big experiment in which I need to remove and preserve a large number of rat brains in one go. Some of these I'm freezing whole for histology and some I wish to preserve in sections/blocks for rna isolation and subsequent PCR analysis. My question is very basic really, what is the best way to freeze these sections? I have had a look around and it generally says snap freeze in liquid nitrogen or on dry ice but I'm not sure what to put the tissue in to do this. Can I just drop it in an eppendorf and then straight into the N2? Should I wrap it in foil (this worries me a bit because the foil may not be free of rnases etc?)? Or should I use melting isopentane (I will be using this for the whole brains anyway)?

Any advice on this matter would help me loads!

h

rather than an ependorf you should use a screw top cell cryo tube (more stable) for liq nitrogen or put it in an eppen and pop it in a -80 freezer

no foil required

dom

Cheers dom, so I just whack the tissue in a tube and drop it into the LN? I appreciate this is probably obvious, just want to be sure!

Helen

yup.

just make sure the lid is on tight (and prob better if tube is dry on outside) - it looks scary but after a couple of goes you lose the fear

have fun

dom

i put the bits and pieces in a screw cap tube (not the cyrotubes for cells... these one's are a bit bigger than that), and into dry ice. it works just as well, and you don't have to worry about freaking out the people on the bench next to you.

V

we dont have access to dry ice (unless its saved from a package - which is rare)

but it seems to make sense (a -80 freezer would probably do the job at a push too)

just pick something which you're sure wont blow up

dom

if you're worried about it not being cold enougn (it is), put a bit of ethanol with the dry ice.

we're lucky, we get it from the hospital supply . though, when we get deliveries with dry ice, someone (and i will find out who), keeps on dumping it in the sink. when i find this person they will be beaten with a soggy noodle. cracked pipes are not fun.

V

We usually have plenty of dry ice, I think I will try that as my dewers will get pretty busy with all the whole brain freezing (hopefully nothing will blow up!).

Thanks guys!