Ambion, Inc.

Transcript Ambion, Inc.

NAT Testing in Solid Tissues and Organs: A Pre-analytical Solution for Nucleic Acid Stabilization

Cindy WalkerPeach, Ph.D.

Ambion Diagnostics Becomes Asuragen!

Ambion Research Products Division Purchased by Applied Biosystems Group (NYSE: ABI) for $273M March 2, 2006 Ambion, Inc.

Formed 1989 Ambion Diagnostics Division Formed 2000 Ambion Services Division Formed 2005 Asuragen, Inc.

Ambion Diagnostics Ambion Services Key Ambion R&D Scientists and Executive Team

RNARetain

™

Pre-analytical Solution • Overview • Applications • Examples • Conclusions

RNARetain Overview

• 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 (RNA

later

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 end user’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

Unmet Clinical Need…………

Easy and Safe Sample Acquisition “Freezes” mRNA and microRNA Expression Profiles Long-Term Cell/ Tissue Banking

Clinical

Virus Preservation Molecular Isolation (RNA/DNA) Convenient Sample Transport

Clinical Research, Tissue/Cell Banking, Expression Profiling Services, Molecular Diagnostics

Intracellular RNA Preservation

5E+5 MCF-7 Breast Tissue Cells

Sample RNARetain 260/280 28S:18S

2.02

1.75

RIN

10.00

Flash Frozen RNARetain Flash Frozen

2.01

1.72

1.87

1.20

1.30

1.45

9.65

9.15

8.90

Flash Freeze RNARetain

Flash Freeze Or RNARetain x4

mir

RNARetain preserves the quality of intracellular RNA to the same extent as the currently established standard of flash-freezing

qRT-PCR Reproducibility

5E+5 MCF-7 Cells In RNARetain 1E+3 HCV1b ARQ Process Control

mir

Vana Extraction HCV1b (process con) and TBP (internal con) qRT-PCR at 40ng sample input x3 x5

Sample # 260/280 28S:18S RIN 1

1.96

1.73

9.87

2 3

1.99

2.09

1.83

1.77

9.97

9.90

4 5

1.98

1.97

1.63

1.77

9.90

9.93

40 45 35 30 25 20 43 41 39 37 35 33 31 29 27 15 25 1 2 3 4 5

Sample Number Reproducible RNA quality and qRT-PCR amplification efficiency is achieved for intracellular RNA extracted from RNARetain preserved cells

Armored RNA

Recovery from RNARetain

1E+7 HCV1b ARQ + or - RNARetain RNA Isolation (Trizol or

mir

Vana Kit) HCV1b qRT-PCR x3 x3 40 35 30 25 20 15

+ + RNARetain

mir

Vana Trizol Process Rep 1 Process Rep 2 Process Rep 3

Pseudo-viral particles can be preserved in and extracted from RNARetain in a reproducible manner

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

Analysis of FNA Specimens

Endoscopic microdissection of pancreatic mass (EU-FNA) Preservation in RNARetain RNA Isolation microRNA expression profiling (qRT-PCR)

Signal ratios for two miRNAs showing differential expression specific to cancerous (ca) or normal (N) tissue are maintained in RNARetain (FNA samples)

6 12 2 8 -2 4 -6 0 -10 -4 -14

Ca FNA

n=7

Ca N Frozen

n=2 n=2

RNARetain preserves miRNA signatures for downstream molecular profiling

-8

Ca Frozen N

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.

Ambion, Inc.

Transcript Ambion, Inc.

NAT Testing in Solid Tissues and Organs: A Pre-analytical Solution for Nucleic Acid Stabilization

Ambion Diagnostics Becomes Asuragen!

RNARetain

Pre-analytical Solution • Overview • Applications • Examples • Conclusions

RNARetain Overview

RNARetain - cGMP Traceability

Selected Publications

Unmet Clinical Need…………

Clinical

Intracellular RNA Preservation

qRT-PCR Reproducibility

Armored RNA

Recovery from RNARetain

RNARetain Data Summary

Analysis of FNA Specimens

Closing Remarks

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