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RecoverAll™ Total
Nucleic Acid Isolation Kit
Recover High Yields of Total Nucleic Acid from Formalin-fixed,
Paraffin-embedded (FFPE) Tissue
For decades, FFPE tissue has been the staple of histologists
and pathologists for creating tissue slides and making patient
diagnoses. More than 400 million FFPE tissue samples exist for
cancer studies alone, and many of these samples can be tied to
clinical data. Using one simple kit, the RecoverAll™ Total
Nucleic Acid Isolation Kit (patent pending), researchers can
now isolate total nucleic acids (DNA and RNA, including miRNA)
from the same FFPE sample. Efficient protease digestion conditions
allow RNA isolation in less than one day. The nucleic acids recovered
are ideal for various downstream applications such as microarray
analysis, qRT-PCR, and mutation screening.
Nucleic Acid Isolation from FFPE Tissue
Ambion has developed a new method for isolating nucleic acids
from human and mouse FFPE tissue. Up to four 20 µm sections
or unsectioned core samples <35 mg can be processed per
reaction. Following deparaffinization and a rigorous protease
digestion, the sample can be split into separate aliquots for
isolation of total RNA (including miRNA) and genomic DNA using
an optimized glass-fiber filter purification protocol.
Total RNA Isolation from FFPE Samples
Although the RecoverAll Kit cannot reverse
RNA fragmentation that may have already occurred (see sidebar,
Challenges of Molecular Analysis of FFPE Tissues), the protease
digestion conditions are designed to release a maximal amount
of RNA of all sizes in a significantly shorter period of time
(3 hr) compared to currently available methods. Recovery of mRNA
has been verified by qRT-PCR (see below), and microRNAs have
been documented with both Northern blot and array analysis (see Examine
MicroRNA Profiles from Archived FFPE Tissue).
Gene Expression from FFPE Samples
Figure 1 shows the results of several real-time RT-PCR experiments
comparing detection of a variety of genes from fixed versus frozen
mouse brain samples. The Ct values from an equal mass of the
FFPE samples were higher than those from the frozen controls,
indicating that other modifications from the fixation process
have adversely affected the RNA as a reverse transcription template.
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Figure
1. Ct Values from Real-Time RT-PCR of
Frozen and FFPE Mouse Brain. Half of each of
four mouse brain samples were flash frozen in liquid
nitrogen then stored at –80ºC; the other half
was fixed and embedded using a standard hospital protocol.
RNA was isolated from one 20 µm slice from each
FFPE mouse brain using the RecoverAll™ Total Nucleic
Acid Isolation Kit. RNA from the frozen controls was
isolated using the mirVana™ miRNA Isolation
Kit. RNA (400 ng) from each sample was used in two-step
real-time RT-PCR. cDNA was synthesized using the RETROscript® Kit.
Random decamer primers, one tenth of the RT reaction,
and SuperTaq™ Polymerase were used for PCR. Each
bar represents the mean ± standard deviation for
8 replicates. TBP=TATA Binding Protein; GAPDH=Glyceraldehyde-3-phosphate
Dehydrogenase; UBC=Ubiquitin C; PKC=Protein Kinase C;
Recc1=Replication Factor C; RNAPol II=RNA Polymerase
II; FAS=Fatty Acid Synthase; DDPK=DNA Activated Protein
Kinase.
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DNA Isolation from FFPE Tissues
Researchers can now recover DNA from FFPE samples just as easily
as RNA by simply increasing the digestion time to 2 days. Isolation
of DNA includes an RNase treatment to fully eliminate RNA. Figure
2 shows an Agilent® 2100 bioanalyzer scan of total nucleic
acids, RNA, and DNA from an FFPE sample processed with
the RecoverAll Kit.
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Figure 2. Agilent® 2100 bioanalyzer Scan Demonstrating Presence of RNA and DNA from FFPE Mouse Liver. Nucleic acids were isolated from FFPE mouse liver (20 µm section, fixed and embedded using standard hospital protocol) using the RecoverAll™ Total Nucleic Acid Isolation Kit. Three equal amounts of sample (based on A260) were treated as follows: untreated control received no DNase or RNase treatment; RNA was isolated by DNase treatment of the nucleic acid sample; DNA was isolated by RNase treatment of the nucleic acid sample. An equal volume of each was analyzed on the Agilent 2100 bioanalyzer. |
Get More Data
from Your FFPE Samples
Many researchers considered their archives of FFPE tissues lost
to molecular analysis. Now, with Ambion's RecoverAll Kit, you
can isolate the full range of nucleic acids from a single FFPE
tissue sample.
Scientific Contributors
Rick Conrad, Tim Barta, Emily Zeringer • Ambion, Inc.
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