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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.

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