Rapid, Easy Extraction of DNA and RNA
from Archival Samples
Judith E. Meis, EPICENTRE Biotechnologies
Introduction
Many attempts have been made to simplify and speed up the extraction of nucleic acids from archival samples.
Traditional protocols require organic reagents to remove paraffin, SDS for cell lysis, and days of overnight proteinase
K digestion followed by further purification.1, 2 In particular, the analysis of nucleic acids from formalin-fixed,
paraffin-embedded (FFPE) tissues is challenging, due to the extensive cross-linking of nucleic acids during the fixation
process. Other challenges include chemical modification and degradation of the nucleic acids, and the limited amount
of nucleic acid in the samples.
The QuickExtract™ FFPE DNA and RNA Extraction Kits provide the simplest and fastest method to reliably produce
PCR-ready DNA and RT-PCR-ready RNA from FFPE tissues. The kits allow nucleic acid amplification from a wide
variety of archival tissue samples without chemical solvents, spin columns, or tube transfers that could result in sample loss.
The QuickExtract FFPE DNA and RNA Extraction Kits are optimized to release the maximum amount of PCR-ready
nucleic acid. Efficient lysis and extraction conditions allow PCR-ready DNA preparation in about an hour, while RNA
extraction takes just over 30 minutes. Extracted DNA can be used in many PCR-based analysis methods including
microsatellite detection, SNP detection, tumor heterogeneity studies, copy number detection, and methylation analysis.
Extracted RNA can be used in both end-point and real-time RT-PCR.
Methods and Results
DNA Extraction and PCR Amplification
Slide-mounted, FFPE-preserved, skeletal
muscle tissue slices (US Biomax, Inc.) were
wet with 100 µl of QuickExtract FFPE DNA
Extraction Solution, scraped off the slide
with a sterile blade, and transferred to the
bottom of small microcentrifuge tubes. The
samples were incubated at 56°C for 1 hour
and then at 98°C for 2 minutes (Fig. 1). The
0.9 cm2 skeletal muscle tissue sample yielded
1.8 µg of extracted DNA as determined by
Hoechst dye fluorimetry. The extracted
DNA was directly amplified with the
FailSafe™ PCR System (EPICENTRE).
Two microliters of extracted DNA was
amplified in an optimized FailSafe PCR
PreMix with a series of primers that detect
regions of three different genes: tumor
protein 53 (TP53), dystrophin (DMD), and
tumor necrosis factor (TNF). The DNA was
denatured at 95°C for 2 minutes, and then
PCR amplified for 40 cycles: 92°C for
15 seconds, 55°C for 15 seconds, and
72°C for 20 seconds. The resulting PCR
amplicons vary from 162 bp to 802 bp in
length, span different regions of the genes,
and demonstrate that these gene targets
(used for tumor heterogeneity studies, and
deletion and insertion mutagenesis studies)
can easily be amplified from DNA extracted
from FFPE samples (Fig. 2).
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www.EpiBio.com PCR-Ready DNA or RNA
Heat at 56°C for
60 min (DNA) or 30 min (RNA)
and then at 98° for 2 min
Fig. 1. Procedure for QuickExtract™ DNA or RNA Extraction from
slide-mounted FFPE Tissue.
M 1 2 3 4 5 6 7 M
bp
– 1000
– 500
– 300
– 200
Fig. 2. PCR amplification of DNA from a slide-mounted, FFPE-preserved
human skeletal muscle tissue section. Following the QuickExtract™ protocol,
2 µl of undiluted, extracted DNA was amplified with primers for three different
loci: TP53, DMD, and TNF. The products were separated on a 3% agarose gel and
were visualized with SYBR® Gold. Lane M, 100-bp DNA ladder; lanes 1-3, exons
2, 3, and 11 of TP53; lanes 4-6, exons 6, 50, and 3 of DMD; lane 7, exon 4 of TNF.
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DNA and RNA Purification
M 1 2 3 4 5 6 7 M
M
1
2
3
4
5
6
7
8
M
bp
bp
– 300
– 500
– 200
– 100
– 200
– 100
Fig. 3. Short Tandem Repeat (STR) PCR amplification of DNA from
slide-mounted, FFPE-preserved, human tissue sections. Following
the QuickExtract™ protocol, 2 µl of undiluted, extracted DNA was
amplified with primers for seven different STR loci. The products
were separated on a 3% agarose gel and were visualized with SYBR®
Gold. The expected allele-to-allele variation in STR sequences results
in multiple products per primer set. Lane M, 100-bp DNA ladder;
lane 1, RENA4; lane 2, D3S1358; lane 3, D7S820; lane 4, THO1; lane 5,
D19S253; lane 6, D21S11; lane 7, AME.
Short Tandem Repeat (STR) PCR amplifications were
performed on DNA extracted from multiple slide-mounted,
FFPE-preserved, human organ tissue sections (Sigma-Aldrich
Co.). The tissue sections were combined and extracted with
the QuickExtract FFPE DNA Extraction Solution as per the
protocol. Two microliters of the undiluted, extracted DNA
was amplified for 40 cycles with primers to seven different
STR loci using the FailSafe PCR System. The DNA was
denatured at 95°C for 2 minutes, and then PCR amplified for
40 cycles: 92°C for 15 seconds, 58°C for 15 seconds, and 72°C
for 20 seconds. All seven regions were successfully amplified
from the extracted DNA (Fig. 3).
[
Gene targets (used for tumor heterogeneity studies, deletion
and insertion mutagenesis studies) can easily be amplified from
DNA extracted from FFPE samples.
]
RNA Extraction and RT-PCR
Slide-mounted, FFPE-preserved, skeletal muscle tissue slices
(US Biomax, Inc.) were wet with 100 µl of QuickExtract FFPE
RNA Extraction Solution, scraped from the slide with a sterile
blade, and transferred to a small microcentrifuge tube. The
samples were incubated at 56°C for 30 minutes and then at
98°C for 2 minutes. The extracted RNA was treated with an
optional DNase I step for 10 minutes at 37°C, stop buffer was
added and the reaction was heated at 65°C for 10 minutes. A
10-µl aliquot of the extracted RNA was reverse transcribed
with random primers into cDNA in a 20-µl reaction using the
MMLV Reverse Transcriptase 1st-Strand cDNA Synthesis Kit
(EPICENTRE).
The cDNA was PCR amplified with primer sets to both
muscle-specific and widely expressed genes. A 3-µl aliquot
Volume 15-1 • May 2008
Fig. 4. RT-PCR amplification of a series of different messages
present in skeletal muscle RNA. RNA was extracted from slidemounted, FFPE human tissue samples per the protocol and reversetranscribed with the MMLV RT 1st-Strand cDNA Synthesis Kit and
random primers. Skeletal muscle cDNA was amplified to produce short
amplicons from a number of different messages. The products were
separated on a 3% agarose gel and were visualized with SYBR® Gold.
Lane M, 100-bp DNA ladder; lane 1, a 116-bp region of RYR1; lane 2,
a 162-bp region of ACTA; lane 3, a 166-bp region of RSP18; lane 4,
a 166-bp region of ENO3; lane 5, a 226-bp region of GAPDH; lane 6,
a 232-bp region of OAZ1; lane 7, a 307-bp region of ACTB; lane 8, a
308-bp region of TNF.
of randomly-primed cDNA was amplified with an optimized
FailSafe PCR PreMix and 10 pmol of each primer. The cDNA
was denatured at 95°C for 2 minutes, and then PCR amplified
for 40 cycles: 92°C for 15 seconds, 55°C-60°C for 15 seconds,
and 72°C for 20 seconds. Eight different messages were easily
detected in the extract with amplicons ranging from 116 bp to
308 bp in length (Fig. 4).
RNA is extensively modified and fragmented during
tissue preservation procedures and storage. The extent of
fragmentation varies depending on the type and length of
fixation as well as the tissue type, but has been reported to
average 200 bases in length.3 Primers were designed to detect
a series of these fragments within one specific message. A 3-µl
aliquot of randomly-primed cDNA was amplified with an
optimized FailSafe PCR PreMix and a series of primers that
detect different regions of the 15.4 kb ryanodine receptor
1 (RYR1) message. The cDNA was denatured at 95°C for
2 minutes, and then PCR amplified for 40 cycles: 92°C for
15 seconds, 58°C for 15 seconds, and 72°C for 20 seconds. The
resulting PCR amplicons spanned the length of the message,
indicating that even though only fragments of RNA are
recovered during extraction, the entire message is represented
in the RNA extract (Fig. 5).
The cDNA produced from DNase-treated, QuickExtract
FFPE RNA is also a suitable template for real-time PCR.
A 1-µl aliquot of skeletal muscle cDNA was amplified
with the TAQurate™ GREEN Real-Time PCR MasterMix
(EPICENTRE) and primers to both β-actin and RYR1. Five
replicates of each reaction, as well as a no-cDNA control
reaction, were performed with 10 pmol of each primer and
the MasterMix. The cDNA was denatured at 95°C for
2 minutes, and then PCR amplified with real-time detection
for 40 cycles: 92°C for 12 seconds, 58°C for 15 seconds, and
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DNA & RNA Purification
1
2
3
4
5
M
Relative Fluorescence Units
M
bp
– 500
– 100
Cycle
Fig 5. PCR amplification of five regions of the 15.4-kb ryanodine
receptor 1 (RYR1) cDNA. RNA was extracted as per the protocol
from FFPE slide-mounted human skeletal muscle tissue samples and
reversed transcribed with the MMLV RT 1st-Strand cDNA Synthesis Kit
and random primers. The RYR1 PCR products were separated on a 3%
agarose gel and were visualized with SYBR® Gold. Lane M, 100-bp DNA
ladder; RYR1 regions amplified: lane 1, 123-228; lane 2, 2,866-2,987; lane
3, 9,132-9,279; lane 4, 11,842-11,979; lane 5, 14,935-15,097.
70°C for 15 seconds. Both messages amplified reproducibly;
data for ACTB are shown in Fig. 6.
[
Even though only fragments of RNA are recovered during
extraction, the entire message is represented in the RNA extract.
]
Conclusions
The QuickExtract FFPE DNA and RNA Extraction Kits
provide a simple, one-tube, rapid DNA or RNA extraction
protocol for PCR- or RT-PCR-based analysis methods.
Once the preserved tissue sample is placed in the extraction
Fig. 6. Successful real-time PCR amplification of skeletal muscle cDNA
produced from extracted RNA. The TAQurate™ GREEN Real-Time PCR
MasterMix was used to detect replicates of two messages in skeletal
muscle cDNA: a 101-bp region of ACTB (red) and a 106-bp region of RYR1
(not shown). The no-template control amplification is shown in black.
solution, heat treatment is all that is required to free the
desired nucleic acid for amplification. No organic extractions,
spin columns, or centrifugation steps are required.
References
1. Dubeau, L. et al. (1986) Cancer Res. 46:2964-2969
2. Goelz, S.E. et al. (1985) Biochem. Biophys. Res. Comm. 130:118-126.
3. Lehmann, U. et al. (2001) Methods 25:409-418.
Ordering Information
QuickExtract™ FFPE RNA Extraction Kit
QFR82805
5 ml (50 rxns)
QFR82050
50 ml (500 rxns)
Includes Extraction Solution and optional DNase Reagents.
QuickExtract™ FFPE DNA Extraction Kit
QEF81805
QEF81050
5 ml (50 rxns)
50 ml (500 rxns)
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Volume 15-1 • May 2008