RNA isolation from large frozen tissue

[stefulj]

I would need to isolate RNA from the frozen mice brains. Unfortunately, someone before has taken the whole brains (about 500mg of tissue) and stored them at -80C.
What would be the safest way (in regard to RNA integrity) to do disruption/homogenisation of so large frozen tissue?
Would it be possible to put frozen brains in RNA-later, and allow them to defrost in order to cut smaller (and specific) region for RNA isolation??
stef

[kiki06]

Hello Stef,
hmm tough question. I guess you could use break off small frozen pieces off the ends if you just need small pieces. Grind to a powder and extract your RNA.

I would not thaw it again, just my opinion.

[stefulj]

Hi Kiki! :-)
I would like to perform gene expression analyses with the isolated RNA. Because different brain regions are heterogeneous in regard to quantitative/qualitative expression of various genes, I would need a specific, defined part of the brain which can't be obtained by breaking off frozen tissue :-(
An alternative is to isolate RNA from the whole organ, but there is that problem with a safe disruption/homogenization...
stef

[kiki06]

Hmm... thats tough. I guess if you need it from a specific organ you have no choice but to thaw it and remove specific region..

Either that or wait until a new mouse arrives and you can do it right next time!

cheers

[rlj22]

I dissect the regions I want from adrenal glands on a glass slide directly on dry ice. I then place a piece of dry ice onto the section I've cut to stop it thawing. Then I quickly stick the pieces into TRI and homogenise. Not had any RNA degradation problems so far! *touch wood*

[shamila]

Hi
How long a tissue may be stored in -70° before RNA extration?my samples are stored since 2000!! would you please help me to know if I still can obtain a good-quality RNA (for QRT-PCR).thanks