Characterization of RNA - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 134

be studied in situ in human tissue (e.g., gene expression in brain
just after stroke or seizure ( 31 )). Optimization of the methods of
RNA extraction are particularly important in this application as brain
tissue is limited in quantity and RNA yields are generally smaller
than many other tissues ( 32 ).

Endogenous RNases must be inactivated immediately upon tissue
harvesting to prevent RNA degradation which can be accomplished
by three accepted methods:

2.2.1. Storage of Brain
Tissue

1. Homogenize samples immediately after harvesting in a
chaotropic-based cell lysis solution (e.g., containing guani-
dinium).
2. Flash freeze samples in liquid nitrogen. To inactivate RNase by
fl ash freezing, it is important that tissue pieces are small enough
to freeze almost immediately upon immersion in liquid nitro-
gen. They must be stored at −80°C and never allowed to thaw.
3. Place samples in RNAlater™ stabilization solution, which is an
aqueous, nontoxic collection reagent that stabilizes and
protects cellular RNA in intact, unfrozen tissue and cell sam-
ples. It is essential that tissue samples are then (0.5 cm) enough
for the RNAlater™ to quickly permeate the tissue. Cells or
tissues can be harvested into RNAlater™ and stored at room
temperature for up to 1 week, at 4°C for up to 1 month, or
at −20°C indefi nitely.

1. Frozen tissue samples should not be allowed to thaw during
handling, shipping, or weighing.
2. Ribonucleases (RNases) are very stable and diffi cult to inacti-
vate. Create and maintain an RNase-free environment: always
wear latex or vinyl gloves, change gloves frequently, do not use
plasticware or glassware without fi rst eliminating possible
RNase contamination, keep tubes closed whenever possible
and use sterile, disposable RNase-free tubes.
3. It is essential to use the correct amount of starting material to
obtain optimal RNA yield and purity.
4. Choose the best RNA isolation method. With all of the differ-
ent RNA isolation methods available, it can be diffi cult to
decide which one to use. The easiest, safest methods available
are column-based methods but they are also more expensive.
Working with a diffi cult tissue, high in fat, a more rigorous,
phenol-based RNA isolation method like ToTALLY RNA™
( www.ambion.com ) or miRNeasy Mini Kit ( www.qiagen.com )
is recommended.
5. Effi cient disruption and homogenization of cells or tissues is
an essential step in RNA isolation that prevents both RNA loss
and RNA degradation. The method of homogenization should

2.2.2. Before RNA
Extraction