RN A I S O L AT I O N
Recover Total Nucleic Acid from FFPE Samples with
Ambion's RecoverAll™ Total Nucleic Acid Isolation Kit
• No overnight Proteinase K digestion required–deparaffinize in the
morning and perform qRT-PCR in the afternoon
Deparaffinization
1. Assemble FFPE sections
equivalent to <
–80 µm or <–35 mg
unsectioned core
• Isolate total nucleic acids, including microRNAs, from FFPE tissue
• Obtain typical yields of >50% that of unfixed tissue from the same
sample source
• Recovered nucleic acids are suitable for qRT-PCR, qPCR,
and mutation screening
• Up to four 20 µm sections or up to 35 mg of unsectioned core samples
can be processed per reaction
Many standard preservation techniques for biological tissue sample storage employ
formaldehyde to maintain tissue structure and prevent putrifecation. However, this type of
preservation makes it difficult to perform molecular analysis on the samples since nucleic
acid is both trapped and modified by significant protein-protein and protein-nucleic acid
crosslinks.
Researchers at Ambion and others (Masuda 1999) have discovered that the fixation
process itself does not necessarily lead to nucleic acid fragmentation. Instead, the
embedding process, which requires high temperatures and vacuum for the paraffin to
penetrate the tissue, quickens chemical reactions which subsequently modify RNA and
DNA. These modifications lead to fragmentation over time.
20 min
3. Centrifuge for 2 min at maximum
speed, and discard the xylene
4. Wash the pellet twice with 1 ml
100% ethanol and air dry
Protease Digestion
1. Add Digestion Buffer and Protease
3 hr or
48 hr
2. Incubate at 50°C for 3 hr for RNA
isolation and 48 hr for DNA isolation
Nucleic Acid Isolation
1. Add 480 µl Isolation Additive
and vortex
2. Add 1.1 ml 100% ethanol and mix
10 min
3. Pass the mixture through a
Filter Cartridge
4. Wash with 700 µl of Wash 1
Deparaffinize in the Morning, Perform qRT-PCR in the Afternoon
The RecoverAll™ Total Nucleic Acid Isolation procedure requires about 45 minutes of
hands-on time and can easily be completed in less than 1 day when isolating RNA. FFPE
samples are deparaffinized using a series of xylene and ethanol washes. Next, they are
subjected to a rigorous protease digestion with an incubation time tailored for recovery
of either RNA or DNA. Nucleic acids are purified using a rapid glass-filter methodology
that includes an on-filter nuclease treatment and are eluted into either water or the low salt
buffer provided.
2. Add 1 ml 100% xylene, mix, and
incubate for 3 min at 50°C
5. Wash with 500 µl of Wash 2/3
Nuclease Digestion and Final Purification
1. Add DNase or RNase mix to
each Filter Cartridge and incubate
for 30 min
45 min
2. Wash with 700 µl of Wash 1
3. Wash twice with 500 µl of Wash 2/3
4. Elute nucleic acid with
2 x 30 µl Elution Solution or
nuclease-free water
RNAi • miRNA • RNA SAMPLE PREPARATION • RNA AMPLIFICATION • RNA RESEARCH PRODUCTS
pfd_RecoverAll.indd 1
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Maximize Yield from Archived and Freshly Fixed Tissue
Ct
20
15
10
5
0
Isolating DNA from FFPE Samples
Although DNA does not fragment as easily as RNA, it is more
sequestered by the formaldehyde treatment, and the DNA isolation
protocol therefore requires a much longer (2 day) protease digestion
time to release substantial amounts of DNA. Recovered DNA can
typically be used for PCR and other downstream applications.
Total Amount of RNA (µg)
RecoverAll
Competitor B
Competitor A
PKC
Recc1 RNAPol II FAS
DDPK
Untreated Nucleic
Acid (Control)
15
12.5
DNase-treated
Nucleic Acid (RNA)
10
7.5
RNase-treated
Nucleic Acid (DNA)
5
2.5
0
14
19
24
29
34
39 44
49
Time (seconds)
54
59
64
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.
8
7
6
5
Brain
4
3
2
1
0
UBC
20
F
Heart
Z
F
Kidney
Z
F
Z
Liver
F
Z
30–
Lung
Colon
Breast
Kidney
Bladder
Yield of RNA from Archived Human FFPE Tissue Samples:
RecoverAll™ vs. Two Competitor Systems
20–
ORDERING INFORMATION
10–
CAT#
SIZE
RecoverAll™ Total Nucleic Acid Isolation Kit
1975
40 purifications
RELATED PRODUCTS
CAT#
SIZE
RNAi
•®
miRNA • RNA SAMPLE PREPARATION
RNaseZap
9780–9784 • RNA
TURBO DNase™
RETROScript®
GAPDH
17.5
Obtain microRNA for Expression Profiling
MicroRNAs (miRNAs) are highly conserved regulatory molecules
expressed in eukaryotic cells. Active, mature miRNAs are 17–24 bases
long and are known to affect the translation or stability of target messenger
RNAs. The nucleic acid recovered using the RecoverAll Kit includes
the full component of miRNA. These miRNA can be used for miRNA
expression profiling when used in combination with the flashPAGE™
Fractionator System and mirVana™ miRNA Array Technology.
TBP
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.
Fluorescence
The ability to isolate nucleic acid that is suitable for molecular analysis
from archived tissue samples provides a powerful tool in retrospective
studies of diseased tissue at both the genomic and gene expression level.
The recovered nucleic acids are suitable for downstream applications
such as endpoint RT-PCR, PCR, qRT-PCR, and mutation screening.
FFPE
25
Gene Expression from FFPE Samples
10
9
Frozen
30
The degree of RNA fragmentation that has already occurred in FFPE
tissues cannot be reversed. However, the nucleic acid isolation of the
RecoverAll Kit are designed to release a maximal amount of RNA
fragments of all sizes, including microRNAs, in a relatively short
amount of time (3 hours).
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.
AMPLIFICATION
• RNA
RESEARCH
PRODUCTS
250 ml – 4 L
2238
1,000 U
Kit
1710
40 rxn
flashPAGE™ Fractionator System
13100
1 each
SuperTaq™ DNA Polymerase
2050
50 U
RT-PCR Grade Water
9935
10 x 1.75 ml
Learn more about
Total Nucleic Acid Isolation Kit at
www.ambion.com/prod/recoverall
RecoverAll™
ORDER ONLINE ANYTIME AT WWW.AMBION.COM
flashPAGE™, mirVana™, RecoverALL™, TURBO DNase™, and SuperTaq™ are trademarks of Ambion, Inc.
RETROscript® and RNaseZap® are registered trademarks of Ambion, Inc.
Agilent® is a registered trademark of Agilent Technologies..
Ambion, Inc.
U.S. 800-888-8804 • Canada 800-445-1161
fax +1-512-651-0201
pfd_RecoverAll.indd 2
Ambion (Europe) Ltd
tel +44 (0)1480-373-020
fax +44 (0)1480-373-010
Ambion K.K. (Japan)
tel +81 (0)3-5638-2181
fax +81 (0)3-5638-2182
For a list of Ambion direct free
phone numbers and distributors
in your country, go to
www.ambion.com/contact
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