RNAlater (R) and collagenase interaction

Go to the profile of cem
Oct 27, 2016
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I am working with a difficult to homogenize tissue, and was wondering if digestion with collagenase would aid in cell separation and lysis. I store my samples in RNAlater or RNAlater ICE as needed. I am working with samples 10-30mg in size and have been frustrated with liquid nitrogen. If I freeze a sample immediately after collection, I have little RNA degradation, but when later isolating, getting an accurate weight is confounded with condensation and ice from liquid nitrogen temperatures.

Does anyone know how collagenase and RNAlater would interact if incubated before or after storing in RNAlater?

2 Comments

Go to the profile of Roberto Rosati
Roberto Rosati 6 months ago

I don't have direct experience on this; however, if you want you can read about the composition of RNAlater in this patent file. Basically RNAlater contains a high concentration of ammonium sulfate. So while the tissues are imbibed in RNAlater, they will be impervious to the action of collagenase; and if you remove RNAlater from the tissue, some RNAse might slowly renature and start degrading RNA... There might be a "sweet spot", but I also don't know if you'll be able to get a satisfactory cell pellet from tissue treated with RNAlater and then disrupted with collagenase. I hope others will comment on this.
About incubating in collagenase before stabilization... I wouldn't risk it, or you'd have to know that this method works for the actual tissue you're working on.
Is cryosectioning an option?

Go to the profile of cem
cem 6 months ago

Took a while but I found a paper that actually preforms this very technique! Read Wasserman, E. Bone 53 (2013) 14-23.