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RNAlater® Tissue
Collection: RNA Stabilization Solution
RNA Remains Stable During Long-term Tissue
Storage
Storage of tissue over extended periods prior
to RNA extraction can degrade RNA resulting in altered gene expression
patterns. The usual answer is to flash-freeze tissue samples
in liquid nitrogen and then store them at –80°C. Although
effective, this technique is problematic and often not practical.
Ambion’s RNAlater® provides a more convenient
method for maintaining tissue for short-term and long-term storage.
It prevents RNA degradation and preserves RNA profiles using
less extreme temperatures. Tissue samples that are stored at
room temperature in RNAlater provide accurate gene expression
results after 72 hours of storage, compared to RNA isolated from
the same samples either immediately after collection or after –80°C
storage for equivalent times (Mutter GL, Zahrieh D, Liu C, Neuberg
D, Finkelstein D, Baker HE, Warrington JA. (2004) Comparison
of frozen and RNAlater solid tissue storage methods
for use in RNA expression microarrays. BMC Genomics 5:88–94.).
But RNAlater also provides much longer storage possibilities.
When keeping the immersed samples at –20°C, RNAlater usually
remains liquid, making the tissue much easier to handle, and
the RNA population remains stable and unchanged for more than
2 1⁄2 years.
RNA From Tissue Stored Long-term
in RNAlater
A long-term storage test was performed on dissected
mouse tissues to examine storage effects on gene expression using
microarray analysis. Dual mice (CD-1 albinos, Harlan Bioproducts)
were sacrificed and heart and brain were split, with one half
quick-frozen and the other placed in RNAlater. Frozen
samples were placed at -80°C and those in RNAlater were
placed at -20°C after several hours at 4°C (per RNAlater Protocol). Samples were stored for 2 years 7 months.
RNA isolation and assessment. RNA
was then prepared using the mirVana™ RNA Isolation
Kit. Yields were equivalent for each tissue, whether frozen or
stored in RNAlater, with heart yielding less RNA than
brain. Analysis on the Agilent® 2100 bioanalyzer provided
RNA Integrity Numbers (RINs) all greater
than 9 (see sidebar, What is RIN?).
Array analysis. For
array analysis, RNA (1 µg) from each heart sample (biological
replicates) was amplified using the MessageAmp™ II procedure
and the cRNA was hybridized to Affymetrix® GeneChips® (Mouse
Genome 430A 2.0). For the brain samples, RNA from only one of
the organs was used; duplicate aliquots were processed to provide
technical replication of amplification and array analysis.
All eight samples generated Present calls of
approximately 60% (Figure 1). There were no consistent trends
in the level of Present calls between the two methods of storage.
Comparison of concordance between samples, shown in Figure 2,
demonstrated a very high correlation of data generated from frozen
and RNAlater samples. Actual concordance values are
provided in Figure 3. For brain, where all samples were from
the same tissue sample, the correlation between frozen and RNAlater replicates
(R=0.996 and 0.997, respectively) was virtually the same as the
average of that between all the possible frozen-RNAlater comparisons
(R=0.994). For the heart samples, where samples 1 and 2 were
from biological replicates, the frozen-RNAlater correlations
(R=0.975 for heart-1 and 0.981 for heart-2) were similar to the
correlation between the frozen (0.976) or RNAlater-treated
(0.984) tissues. More telling, as can be seen in Figure 2, the
core correlation patterns for each brain series overlap, while
the points furthest from the R=1 line do not overlap (i.e., do
not trend in same direction), indicating that there is no systematic
bias introduced by storage in RNAlater. This result
firmly demonstrates that use of RNAlater for extended
storage is equal to the established gold-standard of storage
at -80°C when considering the quality of RNA expression results.
When ease of use and ease of handling of the tissue samples for
RNA extraction are considered, RNAlater is truly superior.
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Figure 1. Percent Present Calls for RNA From Samples Stored Frozen or in RNAlater®. RNA (1 µg) from each heart sample (A. biological replicates) and each brain sample (B. technical replicates) was amplified using the MessageAmp™ II procedure and the cRNA was hybridized to Affymetrix® GeneChips®. Present calls were determined using Affymetrix software. |
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Figure 2. Concordance Comparison
Between Frozen and RNAlater®-stored Samples. Further
analyses of the arrays described in Figure 1 show a comparison
of concordance between samples by overlap for technical
replicates (A) and biological replicates (B).
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Figure 3. Correlations For
Samples Stored Frozen or in RNAlater®.
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Scientific Contributors
Patricia Powers, Rick Conrad • Ambion,
Inc.
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