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Sini SuomalainenApplications Manager
ESHG 2017, May 29
Automated high throughput purification solution for DNA and RNAfor demanding downstream applications
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Precious samples
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Evolution of Cancer Classification
• Cancer classification has changed from anatomical to molecular classification • Molecular subtype–based stratification as a strategy in clinical research
From anatomical to molecular approach
• Breast Cancer• Cervical Cancer• Colorectal Cancer• Liver Cancer• Lung Cancer• Ovarian Cancer• Pancreatic Cancer• Prostate Cancer• Skin Cancer• Stomach Cancer• Thyroid Cancer
• ALK• AKT1• BRAF• EGFR• ERBB2• KRAS• NRAS• MAP2K1• PIK3CA• RET• ROS1• Undefined
Nature Medicine, volume 18, number 3, March 2012
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Advantages
Widespread preserving method
Storage in room temperature
Information on morphology
Advantages
Low cost sampling
Monitoring by multiple sampling
Information on tumor heterogeneity
FFPE Tissue Sample Liquid Sample
Tissue Sampling and Liquid Biopsy
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Challenges
Limited amount available
Variable preparation
Fragmented DNA
Challenges
Low concentration
Preservatives and storage
Genomic DNA
FFPE Tissue Sample Liquid Sample
Tissue Sampling and Liquid Biopsy
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Maximizing efficiency and capacity
Pure nucleic acids are essential for success of monitoring genetic alterations
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Automating Nucleic Acid Isolation
Attach nucleic acids to sil ica surface
Bin d Sample
Remove unwanted impurit ies
Wash Sample
Release purif ied product
Elute SampleSample Lysis
Disr upt cell membranes and
walls
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Maximizing efficiency and capacity
Matched tissue and blood samples
Automated Solution for Isolating Nucleic Acids
MagMAXTM purification reagents KingFisherTM purification system Downstream analysis
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Solution for isolating DNA and RNA from FFPE samples
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MagMAX FFPE DNA/RNA Ultra Kit - DNA and RNA from the same FFPE section
+ DNA Binding Soln
+ Magnetic beads
DNA on beads
RNA in liquid
Wash EluteDNA
RNA, miRNA (and residual DNA) on
beads
RNA, miRNA on
beadsDNase
(optional)
EluteRNA
miRNA
Wash
+ RNA Binding Soln
+ Magnetic beads
Digested FFPE
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Automating cfDNA isolation
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Multiple Sources of Tumor DNA in Blood
• Circulating Tumor Cell (CTC)
2 Main
DNA Targets:
CTCs & cfDNA
• Cell-free DNA (cfDNA)
• Exosomes
Crow ley E, et al. (2013) Liquid biopsy: monitoring cancer genetics in the blood. Nat Rev Clin Onc 10: 472–484.
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• Disrupt cfDNA/histone complexes
• Magnetic particles bind cfDNA fragments
• Wash to remove residual proteins and other impurities
• Release cfDNA in low salt buffer
The Principle of MagMAX™ Cell-Free DNA Isolation
Lysis/Binding
Wash
Elute
LiquidBiopsy™ Blood Collection Kit
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Automating NA purification KingFisher system
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• Large surface = Efficient nucleic acid binding
• Can be used for samples with different viscosities
• Possibility to concentrate the sample
• Easy to automate
• Suitable for low to high throughput applications
Advantages of Magnetic Beads
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• KingFisher instrument family
Powerful KingFisher technology
• Magnetic beads are transferred in the process
Thro
ughp
ut
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User based variability limitations with sample prep
Good Day
Busy Day
Bad Day
KingFisher
KingFisher
KingFisher
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New member in KingFisher family
• KingFisher Presto for high throughput sample purification
• Part of automated system with liquid handling robot
• Reliable and robust processing of magnetic particles
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HamiltonThermo Scientific
Lab Automation
Liquid handling compatibility
Tecan
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Automated purification results
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MagMAX™ FFPE DNA/RNA Ultra Kit
Spin column kit MagMAX - manualMagMAX – automatedwith KingFisher Flex
Spin column kitMagMAX - manualMagMAX – automatedwith KingFisher Flex
DNA
RNA
• Easy to adjust for variable samples• DNA & RNA from same slice• Outperform other methods
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Performance – KingFisher platforms with MagMAX FFPE DNA/RNA
27.7
24.7
26.7
26.1
27.7
24.8
26.8
26.0
2021222324252627282930
1985R 1710L 89-038B 924
Ct
DNA GAPDH g193bp amplicon
Flex (-RT) Presto (-RT)
0
500
1000
1500
2000
2500
3000
1985R 1710L 89-038B 924
To
tal Y
ield
(n
g)
Total DNA Yield (ng)
Flex Presto
Qubit qPCR
DNA
RNA
0
2000
4000
6000
8000
10000
1985R 1710L 89-038B 924
To
tal Y
ield
(n
g)
Total RNA Yield (ng)
Flex Presto
32.2
30.4 34
.1
28.8
33.2
30.1
34.5
28.8
15
20
25
30
35
40
45
1985R 1710L 89-038B 924
Ct
RNA GAPDH g193bp amplicon
Flex (+RT) Presto (+RT)
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MagMAX™ Cell-free DNA Isolation Kit
Red=MagMAX™ cfDNA Blue=Spin column kit
cfDNA peak(150-225 bp)
genomic DNA(>1000 bp)
Enrichment of cfDNA
• Only 3 reagents + Dynabeads®• Ship & store at RT
Convenient
• Room temperature• Lysis & Binding combined• Fast 30-45 min processing time
Minimum effort
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Performance – KingFisher platforms with MagMAX cfDNA Isolation
KF Flex KF Presto
A B C C A A B B C C
2% Agarose GelQubit HS dsDNA
Bioanalyzer HS dsDNA
Donor A Donor B Donor C
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Summary
• KingFisher purification system with MagMAX purification kits offer consistent results from challenging sample types
• Flexibility for different applications• DNA and RNA from same FFPE sample
• Fast and consistent cfDNA isolation
• Eliminated human error in sample and reagent handling
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Thank you for your attention!
For Research Use Only. Not for use for diagnostic procedures.
© 2017 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Bioanalyzer is a trademark of Agilent Technologies, Inc. LiquidBiopsy is a trademark of Cynvenio Biosystems, Inc.
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