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RESOURCE GUIDE
F F P E R N A S a m p l e P reparat i o n R es o u r c e G u i d e
FFPE RNA Sample Preparation Resource Guide
The development of molecular diagnostics, drugs and, ultimately, personalized treatment
for complex diseases such as cancer depends on clinical utilization of robust biomarkers.
Expression profiling using FFPE samples has proven to be a valuable approach for such a
molecular test.
NuGEN’s FFPE RNA sample preparation workflow represents a significant
technological breakthrough, supporting
both retrospective analysis of archived
FFPE tissues for the initial discovery as
well as making it possible to implement
the test in a clinical setting on a routine
basis following current standardized
pathologist practice. This assay is
compatible with a variety of FFPE RNA
isolation kits and supports analysis on
different analytical platforms.
Figure 1
RNA
Extraction
NuGEN’s FFPE RNA Sample Preparation Workflow
RNA
Amplification
qPCR
Sample QC
(optional)
Fragmentation
& Labeling
Genomic
Analysis
NuGEN Tech Rept #1
Encore Biotin Molecule
Affymetrix
3´ Expression Arrays
Fast track your expression biomarker
discovery and diagnostic test development by choosing NuGEN as your
sample preparation partner.
• QIAGEN
• Roche
• Beckman Coulter
Genomics
• Life Technologies
WT-Ovation
FFPE System
WT-Ovation Exon Module
Encore Biotin Molecule
Affymetrix
Exon/Gene Arrays
Encore BiotinIL Molecule
Illumina BeadChips
Unique Features of the NuGEN
FFPE RNA Sample Prep Workflow
NuGEN Agilent App Note
Agilent Expression Arrays
qPCR or other
detection platforms
Simple, robust and reliable
• Starting with 50 ng input FFPE
total RNA
• Compatible with multiple microarray, qPCR and additional detection
platforms
• Automatable
• Highly reproducible
Fast
• Total assay time from total RNA to
array hybridization in one day
Proven
• Customer publications in major
peer-reviewed journals
• Dx partners choosing NuGEN solutions for their test development
cGMP certified
• Expediting regulatory approval path
to molecular diagnostics
Introduction
Formalin fixation coupled with
paraffin-embedding (FFPE) is the most
common method of clinical tissue
sample storage comprising the major
percentage of all archived specimens
in the world. These archives are of
significant value to today’s biomarker
discovery efforts since they are readily
available and are often associated
with in-depth patient clinical records
regarding diagnosis, prognosis, response to treatment and outcome.
In addition, it is a standard procedure
for pathologists to use FFPE samples
for established immunohistochemical
analysis. Consequently, it is highly desirable to utilize the same tissue collection for molecular Dx tests in order to
better correlate the various test results
and reduce the barrier of adoption in
the clinic.
Working with nucleic acids isolated
from FFPE samples, however, has been
challenging. These FFPE samples exhibit marked degradation and change
in response to fixation methods and
long-term storage. Until recently, in
spite of their desirability, these sample
sets have been viewed as unlikely
candidates for global gene expression
analysis using microarrays or large-scale
qPCR analysis.
NuGEN’s unique FFPE RNA Sample
Preparation Workflow takes an innovative approach by employing
the WT-Ovation™ FFPE System for
RNA amplification of formalin-fixed,
paraffin-embedded samples through a
simple, robust and easily automatable
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F F P E R N A S a m p l e P reparat i o n R es o u r c e G u i d e
In addition to providing superior-quality technical results, adopting a new
technology in a clinical setting requires
the tests to pass stringent regulatory
approvals and the process can be
lengthy and time consuming. To help
facilitate and expedite the regulatory
approval process essential for molecular Dx test development, NuGEN has
also implemented rigorous quality programs for reagents manufacturing and
is one of the first sample preparation
companies to receive the cGMP certification. NuGEN’s cGMP certification,
which complements the company’s
ISO 13485 compliance, demonstrates
NuGEN’s quality infrastructure and its
ability to develop and manufacture
highly reproducible genomic sample
preparation products that meet the
“ …microarray analysis is possible,
precise, and reliable from small amounts
of RNA extracted after microdissection
of tumor cells from FFPE tissue
specimens processed for routine
histopathological diagnostics. ”
“ Our approach is feasible and
reliable for a molecular pathological
laboratory in a clinical environment,
as demonstrated by performing the
laboratory protocol at different time
points for different samples and
by applying a practicable
microdissection step… ”
Lassmann, S. et al (2009)
J. Mol. Med, 87:211-224
Figure 2.
cDNA Amplification Yields
10
cDNA Yields (µ) µg
9
8
Array Minimum
solution from as little as 50 ng input of
total RNA. In combination with labeling reagents, the single-day workflow can be tailored for analysis on
the researcher’s detection system of
choice to fit on a variety of microarray
platforms. Alternatively, the amplified
products can be analyzed by qPCR or
a range of other detection platforms,
or archived for future analysis. The
data generated using the NuGEN
workflow are reliable, sensitive, highly
reproducible and biologically relevant
(see the list of selected customer
publications in this document and
available at www.nugeninc.com).
7
6
5
4
3
2 1
3
5
7
9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41
Sample #
41 FFPE-derived Ovarian tumor samples are shown. A minimum of 5.8 µg is required for
analysis on GeneChip arrays (shown by the red bar) a criterion met by all amplifications
in this study.
Ta b l e 1 .
Array Metrics
No. of Arrays
%P
Background
Signal
40
36.3 ± 8.5
28.8 ± 3.1
Scaling Factor cDNA Yield (µg)
26 ± 14
7.8 ± 0.88
Array metrics for the same set of Ovarian FFPE samples shown in Figure 1, obtained from
analysis on the GeneChip HG-U133A Plus 2.0 GeneChip Arrays. % Present calls ranged
from 18-50% for this set of 2- to 15-year-old samples.
premium standards required for diagnostic tests. As a result, NuGEN eases
diagnostic customers’ path to integrating sample preparation into Dx tests
and eventual regulatory approval.
SPIA® - Single Primer Isothermal
Amplification
Central to NuGEN’s FFPE RNA sample
preparation solution is the RiboSPIA technology that provides both
oligo(dT)- and random primer-initiated
amplification. It offers the benefit of
whole transcriptome amplification for
degraded samples. The system yields
sufficient amplified product (cDNA) for
analysis of at least one microarray as
well as qPCR and QC analysis, in about
six hours.
This amplification system can also be
used in archiving strategies where the
amplified cDNA serves as a more stable
source of experimental samples for use
with various analytical platforms and for
sharing among collaborators. This solution’s low input requirement for RNA
allows the preservation and archiving
of the original FFPE block for future
studies.
Highly Robust, Reliable and
Reproducible Array Results
In a collaborative study with the Moffitt
Cancer Center & Research Institute, a
large set of ovarian tumor samples were
amplified and labeled using the NuGEN
FFPE RNA sample preparation workflow
and analyzed on Affymetrix GeneChip®
HG-U133 Plus 2.0 Arrays. The amount
of amplified cDNA recommended for
one array analysis is 5.8 μg, denoted by
a red bar on the histogram in Figure 2.
In this study, sufficient material was
produced for array analysis for all
samples regardless of the age of the
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F F P E R N A S a m p l e P reparat i o n R es o u r c e G u i d e
Array performance averages across
all individuals in this study were also
consistent despite the wide range of
sample ages and the expected variability among different individuals (Table 1).
To demonstrate the technical reproducibility of the workflow, five replicates
were amplified from a three-year old
breast tumor FFPE sample. As shown in
Table 2, a high level of consistency was
obtained with the R2 average of 0.986,
call concordance average of 85.0% and
a %P range of 52.0% – 54.4%.
The NuGEN FFPE RNA sample preparation workflow is robust across a wide
range of samples independent of the
age. It effectively amplifies the FFPE
RNA enabling highly reproducible and
reliable results on microarrays.
Maintaining Biological Significance
from FFPE Samples
To illustrate the integrity of the biological data, RNA extracted from FFPE
tissue was amplified, labeled, hybridized to microarrays and analyzed using
Principal Components Analysis (PCA)
(Figure 3). Targets were prepared
from FFPE RNA from one donor (pink)
and fresh frozen tissue from a second
donor (blue). Each sample was amplified in quadruplicate and hybridized to
GeneChip HG-U133A 2.0 Arrays. This
result indicates a statistically significant
separation of samples based on the tissue disease state for both fresh frozen
and FFPE samples, demonstrating the
amplification system has clearly maintained the biological data integrity.
In an independent study, the differential
expression correlation was assessed
comparing the FFPE samples with the
matching fresh frozen tissues. As shown
in Figure 4, the correlation of differential expression was high, indicating the faithful representation of the
transcriptome using the NuGEN FFPE
RNA sample preparation workflow,
which is extremely critical for biomarker
discovery and diagnostic test development. Similar concordance results
were also obtained on other microarray
Ta b l e 2 .
Array Metrics
A.
Signal R2
1
2
0.985
3
0.983
0.986
4
0.981
0.987
0.991
5
0.987
0.989
0.987
0.986
% Call
Concordance
1
2
3
4
2
84.6
3
85.3
84.4
4
84.8
84.5
85.0
5
85.7
85.0
85.5
2
3
4
B.
85.4
C.
Array Metrics
SF
Bkgd
%P
GAP
Act
RawQ
Average
6.8
29.0
53%
0.8
12.0
0.8
SD
0.58
1.13
1.1%
0.03
0.48
0.05
Five replicate amplifications performed on 50 ng of total RNA derived from a three-year
old breast tumor FFPE sample were analyzed on GeneChip arrays. Results show highly reproducible and robust array performance. Signal R2 and Call Concordance shown in panels
A and B have an average of 0.986 and 85.0%, respectively. The %P shown in panel C was in
a range of 52.0% – 54.4%.
Figure 3.
Principal Components Analysis (PCA) of Colon Tumor and
Normal Adjacent Tissue (NAT).
60
48
NAT
36
24
PCA #2
samples, indicating the robustness of
the amplification technology.
12
0
-12
-24
Tumor
-36
-48
-60
-230 -184 -139
-94
-49
-4
41
86
132
177 222
PCA #1
Targets were prepared from RNA extracted from formalin-fixed, paraffin-embedded tissue (FFPE) from one donor (red) and fresh frozen tissue from a second donor (blue). Each
sample was amplified in quadruplicate and hybridized to Affymetrix GeneChip HG-U133A
2.0 Arrays. PCA was performed using Partek Genomics Suite software. The green and
blue ellipses (NAT and Tumor, respectively) define the boundary of 2 standard deviations
from the centroid of each cluster, indicating a statistically significant separation of samples
based on the disease state of the tissue. This demonstrates that the amplification systems
maintain the integrity of the biological data.
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F F P E R N A S a m p l e P reparat i o n R es o u r c e G u i d e
platforms such as Affymetrix GeneChip
Exon/Gene Arrays, Illumina BeadChips
and Agilent Whole Genome Expression
Arrays (data not shown).
Figure 4.
Highly Concordant Differential Expression Analysis Results Between
FF and FFPE Samples
Signal Log Ratio: FFPE vs. FF Signal Log Ratios (P>.0001, N=815)
3
Unique qPCR-based Sample QC
Method for Sample Qualification
FF SLR
Due to the expected wide range of
quality and integrity for FFPE-derived
RNA, there is a pressing need for tools
that allow assessment of sample quality
so researchers can determine the level
of performance expected with the
samples under study. The more tradi-
2
y = 1.2x - 0.042
R2= 0.92
1
0
-2.5
-2
-1.5
-1
-0.5
2
2.5
-3
Using the WT-Ovation FFPE System, total RNA isolated from lung cancer and normal FFPE
samples (50 ng) as well as matching fresh frozen (FF) tissues (2 ng) were amplified, labeled
and hybridized to Affymetrix GeneChip® HG-U133A Arrays. 815 genes were detected to
be highly differentially expressed (signal log ratio, SLR) between the cancer and normal
samples. Highly concordant results are demonstrated between FF and FFPE samples.
Figure 5.
RNA Quality Assessment of qPCR Assay
A
60
R2 = 0.97
50
R2 = 0.75
40
R2 = 0.82
30
20
10
0
0
Briefly, the NuGEN FFPE sample QC
method was developed after surveying a panel of amplicons from housekeeping genes across diverse tissues
and comparing their expression level
normalized to a reference RNA sample
(HeLa RNA). Amplicons that correspond to portions of the beta actin and
1.5
FFPE SLR
Tanney, A. and Kennedy, RD. (2010)
Personalized Medicine, 7(2):205-211
2
4
6
8
10
12
gACTB delta Ct (FFPE - HeLa)
B
60
R2 = 0.99
50
R2 = 0.79
40
R2 = 0.89
%P
tional method of correlating Bioanalyzer trace or sample age with microarray
expression analysis outcome has been
shown to be ineffective. As a result,
NuGEN has developed a new sample
QC method immediately following
reverse transcription and second-strand
cDNA synthesis of RNA extracted from
FFPE blocks that are predictive of the
performance of the microarray assay in
a given sample set.
1
-2
%P
“ In the future, the use of molecular
subtyping and companion diagnostics
to guide patient care will become
standard practice. FFPE remains the
standard method for sample storage in
routine clinical practice. The ability to
work with FFPE samples will therefore
be a key component to biomarker
discovery, validation and, ultimately,
delivery in the clinic. ”
0.5
-1
“ In our own experience, it has been
possible to extract good quality
transcriptional data from samples as old
as 17 years using the Roche High Pure
FFPE extraction kit and the NuGEN
WT-Ovation FFPE system. ”
0
30
20
10
0
-5
0
5
10
15
18S delta Ct (FFPE - HeLa)
Delta Cts between FFPE and control RNAs show high correlation to array performance (%P).
Delta Cts for three different sample sets were calculated and plotted versus the %P for the
corresponding arrays. A correlation for the gACTB amplicon is shown in Figure 5A and for
the 18S rRNA amplicon in Figure 5B. Blue represents a 21-sample lymphoma set, green
represents a 40-sample ovarian tumor set and yellow represents a 2-sample breast tumor
set (with 5 replicates of each plotted).
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F F P E R N A S a m p l e P reparat i o n R es o u r c e G u i d e
18S rRNA transcripts were selected
because they exhibited stable expression pattern across multiple tissues
and high correlation to array performance, as measured by %Present call
on Affymetrix GeneChip U133 Arrays
in the 63 FFPE RNAs tested (Figure 5).
Detailed design and sequences of the
amplicons are described in the NuGEN
WT-Ovation FFPE v2 Technical Note #1
(download at www.nugeninc.com).
This novel RNA sample quality assessment assay correlates qPCR results
to the quality of microarray results
generated with these samples. This
in-process quality assessment assay, in
conjunction with a pilot study approach
described in the FFPE Validation Guidelines (NuGEN Bulletin 9, download at
www.nugeninc.com), can predict the
performance of a given set of FFPE
samples, allowing researchers to utilize
these valuable samples to their fullest
potential.
“ With several commercially available
kits for nucleic acid extraction that
appear to work equally well, and a
robust RNA amplification kit such as
the NuGEN Ovation FFPE System, we
achieved functional, reproducible, and
reliable microarray data from FFPE
material from multiple tissues. ”
Abdueva, D. et al (2010)
J. Mol. Diagnostics, 12(4):409-417
Conclusions
Expression profiling from FFPE samples
has been recognized to play a pivotal
role in discovery of biomarkers and
development of molecular diagnostic
tests for complex diseases such as cancer. To meet the challenges of working with this sample type that is both
important for retrospective studies and
standard practice in established pathology setting, NuGEN has developed
a robust, reliable, and flexible sample
preparation workflow. Since NuGEN
has also established rigorous manufacturing processes (achieving both ISO
13485 and cGMP certification). These
methods can be used to generate
reproducible, biologically significant
data for signature development and
use in a clinical setting. These qualifications make it easier and faster for
the diagnostic partners to integrate
NuGEN reagents as a part of their Dx
tests expediting the path to regulatory
approval.
Fast track your expression biomarker
discovery and diagnostic test development by choosing NuGEN as your
sample preparation partner.
NuGEN WT-Ovation FFPE System
Technical Report #2 – The WT-Ovation
FFPE System Performance
NuGEN Technologies Bulletin 9 –
WT-Ovation FFPE System Validation
Guidelines and the RNA Sample Quality Assessment Tool Pilot Study
Selected Customer Publications
(For a more complete list of
publications and web seminars, visit
www.nugeninc.com)
Lassmann, S. et al (2009) J. Mol. Med,
87:211-224
Abdueva, D. et al (2010) J. Mol.
Diagnostics, 12(4):409-417
Tanney, A. and Kennedy, RD. (2010)
Personalized Medicine, 7(2):205-211
Customer Support
NuGEN provides worldwide technical
support. To learn more, call us at (888)
654 6544 or (650) 590 3600, or visit us
on the web at www.nugeninc.com.
Ordering Information
Related References
Part No.
Product Name
NuGEN Agilent Application Note
3400
WT-Ovation™ FFPE System V2
4200
Encore™ Biotin Module
4210
Encore™ BiotinIL Module
2000
WT-Ovation™ Exon Module
NuGEN WT-Ovation FFPE System
Technical Report #1 – RNA Sample
Quality Assessment Test for the WTOvation FFPE System
NuGEN Technologies, Inc.
Headquarters USA
Europe
201 Industrial Road, Suite 310
San Carlos, CA 94070 USA
Toll Free Tel: 888.654.6544
Toll Free Fax: 888.296.6544
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The Netherlands
Tel: +31-13-5780215
Fax: +31-13-5780216
[email protected]
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information, visit our website
www.nugeninc.com
©2010 NuGEN Technologies, Inc. All rights reserved. The Ovation® and Applause™ families of products and methods are covered by U.S.
Patent Nos. 6,692,918, 6,251,639, 6,946,251 and 7,354,717, and other issued and pending patents in the U.S. and other countries. NuGEN,
the NuGEN logo, Ovation, SPIA, Ribo-SPIA, WT-Ovation, Applause, Encore, Prelude and Imagine More From Less are trademarks or registered trademarks of NuGEN Technologies, Inc. Other marks appearing in these materials are marks of their respective owners.
For research use only.
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