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Examine MicroRNA Profiles from Archived Formalin-fixed, Paraffin-embedded
(FFPE) Tissue
Ambion’s new RecoverAll™ Total Nucleic
Acid Isolation Kit (patent pending) is designed for quantitative
recovery of RNA and/or DNA from FFPE samples (see Recover
High Yields of Total Nucleic Acid from FFPE Tissue).
The nucleic acid from the FFPE samples includes the full complement
of microRNA (miRNA). These miRNAs can be used for miRNA expression
profiling when used in combination with the flashPAGE™ Fractionator
System and mirVana™ miRNA Array Technology (see Getting
Started with MicroRNA Research).
The mirVana miRNA Labeling Kit and mirVana
miRNA Probe Set were used to analyze microRNA (miRNA) expression
profiles in RNA preparations from formalin-fixed, paraffin-embedded
(FFPE) mouse tissues. To confirm the accuracy of the miRNA expression
results, the miRNA expression profile obtained from each FFPE
sample was compared to that of a matched flash-frozen sample.
Isolate microRNA from FFPE Samples
Kidney, liver, and brain tissues from 4 mice were harvested,
and half of each tissue was immediately flash-frozen, while the
other half was formalin-fixed and paraffin-embedded following
a procedure commonly used in hospitals [1]. Total RNA was isolated
from frozen tissues using the mirVana miRNA Isolation
Kit and from FFPE samples using the RecoverAll Total Nucleic
Acid Isolation Kit, which is partly based on mirVana
technology. miRNA was isolated and concentrated from each sample
using the flashPAGE Fractionator and the flashPAGE Clean-up Kit.
Total RNA (1 µg) from representative tissue samples was
also analyzed on a Northern blot hybridized with a probe specific
for let-7 (Figure 1). The recovery and quality of the let-7 miRNA
in the FFPE and frozen samples were very similar, indicating
that the new FFPE RNA isolation procedure was effective in recovering
intact miRNA.
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Figure
1. let-7 miRNA from FFPE and Flash-frozen
Mouse Tissue Samples. Total RNA was isolated
from four different mouse tissues that had been frozen
(F) or formalin-fixed and paraffin-embedded (Z). This
Northern blot (1 µg RNA/sample) was incubated with
an oligonucleotide probe specific for let-7 and verified
the presence of intact let-7 miRNA.
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Obtain Reproducible miRNA Array Results
A pool containing miRNA from 5 different tissues was created
as a means of comparing individual samples to each other. Using
the mirVana miRNA Labeling Kit, miRNA from the pooled
sample was labeled with Cy™3, and miRNA from individual FFPE
or frozen tissue samples was labeled with Cy5. Each Cy5-labeled
miRNA sample was co-hybridized on a separate miRNA array with
a portion of the Cy3-labeled pooled miRNA sample. The relative
Cy dye signal intensities were compared for each miRNA, and hierarchical
clustering showed that miRNA profiles were nearly identical between
FFPE and frozen tissues (Figure 2). Correlations of 93.5%, 95.9%,
and 97.8% were obtained for kidney, liver, and brain tissues,
respectively, by comparing the average Cy5/Cy3 (R/G) ratio of
the fixed samples to that of the frozen samples (Figure 2).
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Figure
2. Excellent Correlation of miRNA Expression
Profiles from FFPE and Flash-frozen Samples. For
each tissue type, the R/G Normalized (Mean) ratio of
each set of 4 fixed samples and 4 frozen samples was
averaged. The average R/G ratio of the FFPE sample (X
axis) was plotted against the average R/G ratio of the
frozen sample (Y axis) to determine the correlation between
miRNA profiles generated from fixed tissue samples as
compared to frozen tissue samples (A. Kidney, B. Liver, C. Brain). D. Representative
heat maps from fixed (FFPE, left) and frozen (right)
samples from brain.
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Detect Differentially Expressed miRNAs
The goal for most miRNA array analyses is to identify the miRNAs
that are differentially expressed between samples. To confirm
that cross-comparisons of FFPE samples provide the same differential
expression data as frozen samples, we compared miRNA array data
from both the FFPE and frozen samples for kidney and brain tissues.
The expression ratio of the FFPE kidney versus FFPE brain was
compared to the expression ratio of the frozen kidney versus
frozen brain (Figure 3). The correlation between the FFPE tissue
comparison and frozen tissue comparison exceeded 95%. The list
of miRNAs that are estimated to be two-fold differentially expressed
between kidney and brain samples was very similar for both frozen
and FFPE sample comparisons (Figure 3).
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Figure 3. Similar
Set of miRNAs Were Found to Be Differentially Expressed
in Different Tissues in Fixed and Frozen Samples. The
R/G Normalized (Mean) ratio of each set (n=4) of fixed
and frozen samples was averaged for kidney and brain
tissues. Panel A. The kidney to brain
expression ratio for both fixed and frozen samples was
determined, and the expression ratio of the fixed kidney
vs. fixed brain (X axis) was plotted against the expression
ratio of the frozen kidney vs. frozen brain (Y axis). Panel
B. Representative examples of miRNA differentially
express by at least 2-fold in kidney vs. brain are shown.
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Examine Global
miRNA Expression in FFPE Tissues
Our data show that miRNA profiles are maintained and are not
significantly affected by the fixation process. This introduces
a significant opportunity for using archived samples to detect
changes in miRNA expression levels in various disease states,
with ramifications for defining regulatory roles of miRNA in
biological processes.
Scientific Contributors
Emily Zeringer, Patricia Powers, Jaclyn Shingara, Kerri Keiger,
Tim Barta, Rick Conrad, David Brown • Ambion, Inc.
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