NAT Testing in Solid Tissues
and Organs: A Pre-analytical
Solution for Nucleic Acid
Stabilization
Cindy WalkerPeach, Ph.D.
cwalkerpeach@asuragen.com
Copyright © 2007 Asuragen, Inc
Ambion Diagnostics Becomes Asuragen!
Ambion, Inc.
Formed 1989
Ambion
Research Products
Division
Purchased by Applied
Biosystems Group
(NYSE: ABI) for $273M
March 2, 2006
Copyright © 2007 Asuragen, Inc
Ambion Diagnostics
Division
Ambion Services
Division
Formed 2000
Formed 2005
Asuragen, Inc.
Ambion Diagnostics
Ambion Services
Key Ambion R&D Scientists
and Executive Team
RNARetain™ Pre-analytical Solution
• Overview
• Applications
• Examples
• Conclusions
Copyright © 2007 Asuragen, Inc
RNARetain Overview
Copyright © 2007 Asuragen, Inc
•
Versatile, non-toxic, non-organic solution for
preservation of nucleic acids by inactivating nucleases
to prevent degradation of RNA/DNA
•
Stabilizes nucleic acids at room temperature, without
requirement of dry ice or liquid nitrogen, thereby
allowing biological samples to be shipped on wet ice or
at ambient temperatures
•
Compatible with all downstream RNA extraction
methods
•
Based on formulation widely used in life science
research (RNAlater - Ambion, Inc.)
•
Covered by US patent: 6,204,375 and WW equivalents
RNARetain - cGMP Traceability
• Manufactured under QSR 21 CFR Part 820 guidelines to ensure
lot-to-lot consistency critical for reagents used in clinical
specimen collection and storage for downstream RNA extraction
and molecular analysis
• Compatibility with major extraction procedures and molecular
analysis techniques will allow end-user to qualify reagent for use
in specific downstream applications
• cGMP traceability provides consistent performance in enduser’s hands
Copyright © 2007 Asuragen, Inc
Selected Publications
• Development and validation of a method for using breast core
needle biopsies for gene expression microrarray analyses
Matthew Ellis, et al. Clin Cancer Res. 2002, 8:1155-1166
• The effect of sample degradation and RNA stabilization on
classical swine fever virus RT-PCR and ELISA methods
Stuart D. Blacksell, Syseng Khounsy and Harvey A. Westbury.
J. Vir Methods. 2004, 118(1):33-37
• Viral infectivity is maintained by an RNA protection buffer
C. Uhlenhaut and M. Kracht. J. Virol Methods. 2005, 128(1-2):189-91
• Prognostic gene expression signatures can be measured in
tissues collected in RNAlater preservative
Dondapati Chowdary, et al. JMD. 2006, Vol. 8, No. 1
• Currently there are over 1,000 citations since Jan 2001 in
HighWire Press: http://Highwire.Stanford.edu
Copyright © 2007 Asuragen, Inc
Unmet Clinical Need…………
Easy and Safe
Sample Acquisition
Long-Term Cell/
Tissue Banking
“Freezes” mRNA and microRNA
Expression Profiles
Clinical
Molecular Isolation
(RNA/DNA)
Virus Preservation
Convenient
Sample Transport
Clinical Research, Tissue/Cell Banking, Expression Profiling Services, Molecular Diagnostics
Copyright © 2007 Asuragen, Inc
Intracellular RNA Preservation
mirVana
Sample
Trizol
5E+5 MCF-7
Breast Tissue Cells
2.02
1.75
10.00
Flash Frozen
2.01
1.20
9.65
RNARetain
1.72
1.30
9.15
Flash Frozen
1.87
1.45
8.90
Nanodrop®
Agilent Bioanalyzer
Copyright © 2007 Asuragen, Inc
mirVana
RNARetain
Trizol
mirVana™ or Trizol®
Extraction
x4
RIN
RNARetain
Flash Freeze
Flash Freeze
Or RNARetain
260/280 28S:18S
RNARetain preserves the quality of intracellular
RNA to the same extent as the currently established
standard of flash-freezing
qRT-PCR Reproducibility
x5
40
1E+3 HCV1b ARQ
Process Control
45
43
35
41
TBP Median Ct
39
mirVana
Extraction
30
37
35
25
33
31
20
HCV1b Median Ct
5E+5 MCF-7 Cells
In RNARetain
Sample # 260/280 28S:18S RIN
1.96
1.73
9.87
1
1.99
1.83
9.97
2
2.09
1.77
9.90
3
1.98
1.63
9.90
4
1.97
1.77
9.93
5
29
27
HCV1b (process con)
and TBP (internal con)
qRT-PCR at 40ng
sample input
Copyright © 2007 Asuragen, Inc
15
25
1
x3
2
3
4
5
Sample Number
Reproducible RNA quality and qRT-PCR
amplification efficiency is achieved for
intracellular RNA extracted from RNARetain
preserved cells
TBP
ARQ
HCV1b
Armored RNA® Recovery from RNARetain
1E+7 HCV1b ARQ
+ or - RNARetain
40
Median Ct
35
RNA Isolation
(Trizol or mirVana Kit)
x3
30
25
20
15
+
-
mirVana
HCV1b qRT-PCR
x3
Process Rep 1
+
-
RNARetain
Trizol
Process Rep 2
Process Rep 3
Pseudo-viral particles can be preserved in and
extracted from RNARetain in a reproducible manner
Copyright © 2007 Asuragen, Inc
RNARetain Data Summary
• Preserves quality of intracellular RNA to same extent
as flash-freezing
• Does not interfere with reproducibility of intracellular
RNA extraction and analysis methods
• Effective for storage and preservation of viral RNA as
indicated by compatibility with Armored RNA
Copyright © 2007 Asuragen, Inc
Case
Study:
C
p=1.77E-13
14
12
10
miRNAs
6
4
2
0
Normal
Chronic
Cancer
Cell Line
196Ct - 217Ct
8
10
8
6
4
2
0
-2
-4
-6
-8
N
N
Ca Ch
Ca Ch
n=7 n=10 n=7
Combination of two microRNAs can be used to distinguish normal,
chronic pancreatitis, and carcinoma samples
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Analysis of FNA Specimens
Signal ratios for two miRNAs showing differential
expression specific to cancerous (ca) or normal (N)
tissue are maintained in RNARetain (FNA samples)
Endoscopic microdissection
of pancreatic mass
(EU-FNA)
6
RNA Isolation
2
196aCt- 217Ct
Preservation
in RNARetain
196aCt- 217Ct
12
-2
-6
Copyright © 2007 Asuragen, Inc
4
0
-10
-4
-14
-8
Ca
FNA
n=7
microRNA expression
profiling (qRT-PCR)
8
Ca
N
Frozen
n=2
n=2
RNARetain preserves miRNA
signatures for downstream
molecular profiling
Ca
N
Frozen
n=10
n=7
Frozen tissues from
Szafranska et al.
Closing Remarks
•
RNARetain is a pre-analytical solution which stabilizes intracellular RNA in
cells and solid tissues for downstream extraction and molecular
characterization
•
RNARetain is compatible with Armored RNA, illustrating potential utility in
collection and storage of viral-infected tissues, thus eliminating the need for
immediate processing or flash freezing
•
Next step will be to identify beta test sites to evaluate clinical utility of
RNARetain for collection and transport of viral-infected tissue specimens
•
Agendia, B.V.
Used in conjunction with MammaPrint®
70 gene expression signature which predicts the risk
of breast cancer recurrence
Received US FDA approval Feb 6, 2007
Copyright © 2007 Asuragen, Inc