|
RNAlater® for
RNA and Protein Stabilization
•Simplify sample
collection
•No need to freeze
sample in liquid nitrogen
•Stabilize both
RNA and protein within tissues or cells
•Isolate both RNA
and protein from the same sample
RNAlater is an aqueous,
nontoxic, tissue and cell collection reagent that stabilizes
and protects RNA in intact, unfrozen tissue and cell samples.
The solution permeates the cells, stabilizing the RNA inside
them. RNAlater treated samples can be
stored at room temperature (25°C) for up to one week,
at 4°C for up to one month, or at -20°C (or
lower) indefinitely. Intact protein can be recovered from RNAlater-stabilized
samples for use in downstream applications such as Western
blotting or 2D gel analysis. Thus, RNAlater is
the ideal companion to the PARIS™ (Protein
And RNA Isolation System) Kit, a kit specifically engineered
for the isolation of both protein and RNA from the same experimental
sample. (See "RNAi: Get the Whole Story")
To demonstrate the utility of RNAlater for
preserving proteins and RNA for long periods of time, RNA and
protein were isolated from mouse tissue that had been stored
for more than a year in RNAlater. Fresh brain and kidney
tissues were dissected and immediately submerged in 5 volumes
of RNAlater, incubated at 4°C overnight and then
transferred to -20°C. Thirteen months later, the
RNAlater treated samples were thawed at room temperature,
about 50 mg of tissue was collected, and the remainder of the
samples were refrozen. Small tissue pieces from both tissues
were subjected to the standard PARIS procedure
to quickly isolate total RNA and protein. The samples were
disrupted with a motorized homogenizer in 400 µl of Cell
Disruption Buffer. The bulk of the resulting lysate (300 µl)
was then immediately mixed with 1 volume of 2X Lysis/Binding
Solution for RNA isolation using the glass fiber filter provided
with the PARIS Kit, and the remainder of the lysate was reserved
for protein analysis.
|
Figure 1. RNA
and Protein from Tissues Stored in RNAlater® for >1
Year. RNA and
protein were isolated with the PARIS™ Kit from
~50 mg of mouse brain or kidney stored for 13 months
at -20°C in RNAlater. Purified RNA
(1 µg) was analyzed on a denaturing agarose gel
and subsequently transferred, blotted, and hybridized
using the NorthernMax®-Gly Kit (Ambion). A portion
of the protein fraction (10 µg) was used for Western
blot analysis with antibodies specific for GAPDH or
Hur proteins. |
|
Figure 2. RNA
and Protein Isolation from Stabilized Samples. Isolation
from ~30 mg of mouse liver, brain or kidney 5 days
after collection. Samples were either kept frozen at
-80ºC,
or stored in RNAlater® at 4ºC. |
As shown in Figure
1, no obvious RNA degradation was observed by denaturing agarose
gel analysis or by Northern blot hybridized with probes specific
for U1 snRNA, or ß-actin and GAPDH mRNAs. Analysis of
the protein fraction by Western blot showed that the recovered
proteins were full-length. (Note: The multiple bands observed
in the Hur blot correspond to brain-specific higher molecular
weight isoforms of the Hur protein.) Figure 2 further confirms
that protein yield and quality are not affected by RNAlater treatment.
These results demonstrate that RNAlater is an efficient
sample collection/stabilization reagent for protecting RNA,
and that RNAlater-treated samples can be used for routine protein
analysis such as Western blotting.
It is especially useful
for the following applications:
•Archiving samples for future analysis
•Collecting samples at different
time points without having to
process them immediately
•Shipping samples without dry ice
•Collecting samples in the field
back
to top
|
