Thaw
Frozen Tissues
Without Damaging
RNA
• Process
previously frozen tissues like freshly harvested samples
• Thawing
tissues in RNAlater®-ICE
protects RNA from degradation
• No
more tissue pulverizing with mortar and pestle and awkward
transfer of powder to tube
• Easily
apportion frozen tissue samples for multiple experiments
RNAlater-ICE Frozen Tissue Transition Solution
is a unique RNA stabilizing solution. Simply drop frozen tissues
into RNAlater-ICE and walk away! Once tissues are thawed
they can be easily processed using standard RNA isolation procedures.
No more laborious grinding of frozen tissue to preserve RNA in
difficult tissues or tissues that need to be stored prior to
isolation. Treated tissues can be directly inserted into standard
homogenization and isolation protocols and processed as though
fresh.
Quick Freezing Tissues Preserves RNA
Historically, tissues that need to be
stored prior to RNA isolation are "snap" or "flash" frozen on dry ice or in
liquid nitrogen to preserve RNA integrity. RNA in tissue is stable
while frozen at -80ºC but thawing the tissue prior to or
during its disruption can result in RNA degradation. This is
true even if the tissue thaws while in the denaturation solution.
Processing Frozen Tissues is Problematic
Frozen tissues are typically ground with a chilled
mortar and pestle for quality RNA isolation. Liquid nitrogen
must be added to the mortar to keep the sample frozen while it
is ground. For multiple samples this process is laborious. Either
a separate mortar and pestle set is needed for each sample, or
the set must be thawed and cleaned after each sample is processed
to avoid cross-contamination. Powdered tissue can also thaw during
transfer to a homogenization vessel. This often results in formation
of clumps that do not readily disperse in the lysis solution,
resulting in RNA degradation and loss. Very small samples should
be homogenized immediately in lysis solution, which can
again be cumbersome if there are multiple samples to process.
Process Frozen Tissue Without
Jeopardizing RNA Integrity — RNAlater-ICE
RNAlater-ICE solves all of these problems.
Simply submerge frozen tissue samples in 10 volumes of RNAlater-ICE
and store overnight at
-20 or -80ºC (the solution will remain liquid at these temperatures).
As the tissue thaws, RNA integrity is protected. Once treated, tissue can be
safely stored at 4ºC or even at room temperature (for a limited period
of time) and can be further dissected or processed prior to homogenization
in a standard RNA isolation lysis buffer. Thus the same frozen tissue sample
can be used multiple times for different experiments without compromising RNA
integrity.
Figure 1 shows the quality
of RNA isolated from three different frozen mouse tissues that
were immediately homogenized
or thawed in RNAlater-ICE overnight at -20ºC. Both
the stained gel and the resulting Northern blot demonstrate that
RNAs isolated from tissues treated with RNAlater-ICE maintain
a high degree of integrity. The dramatic preservation of RNA
by RNAlater-ICE is also illustrated in Figure 2. Here
we compare RNA that was prepared directly by homogenization of
frozen tissue, with a sample that was allowed to thaw at room
temperature for 5 minutes,
and a sample that was frozen,
soaked overnight at -20ºC in RNAlater-ICE, and kept at room temperature
for 30 minutes before being processed for RNA isolation. The protective effect
of RNAlater-ICE is obvious.
|
|
| Figure 1. Quality
of RNA From Samples Treated With RNAlater-ICE. Total
RNA was isolated from various frozen mouse tissue samples
that were either processed directly from a frozen state
or thawed in RNAlater-ICE at -20ºC overnight. (A) shows
the ethidium bromide stained RNA in a denaturing agarose
gel. (B) shows the results of Northern blot analysis
of the same gel hybridized to radiolabeled probes for ß-actin,
GAPDH, and cyclophilin. (Note that the specific activity
of the cyclophilin probe was lower than that of the other
probes.) For each tissue the integrity of both rRNA
and mRNAs for the RNAlaterICE treated samples
is comparable to that of RNA prepared directly from frozen
tissue samples. |
|
|
| Figure 2. RNA
Integrity from Frozen Samples Ground, Thawed or Treated
with RNAlater-ICE. Total
RNA was isolated from mouse liver samples that were
processed directly from a frozen state (ground), thawed
on a benchtop for 5 min (thawed), or thawed overnight
at -20ºC in RNAlater-ICE and then stored
at rt for 30 min prior to RNA isolation (treated). (A) shows
the ethidium bromide stained RNA in a denaturing agarose
gel. (B) shows the results of Northern blot
analysis of the same gel hybridized to radiolabeled
probes for ß-actin, GAPDH, and cyclophilin. Note
that frozen tissue thawed in the absence of RNAlater-ICE
yielded degraded RNA while RNA remained intact when
tissue was thawed in RNAlater-ICE. |
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