Improved workflow for FFPE tissue Nucleic Acid...
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Guillaume Durin Edwin Rudd Gary Smejkal John-David Herlihy Jim Laugharn Covaris, Inc. • 14 Gill Street, Unit H, Woburn, Massachusetts 01801-1721 USA Tel: +1 781-932-3959 • Fax: +1 781-932-8705 • Email: [email protected] Web: www.covarisinc.com Improved workflow for FFPE tissue Nucleic Acid extraction Paraffin removal – Emulsification without organic solvent Seamless Integration with NGS applications – High throughput capable Tissue samples are commonly preserved by Formalin Fixation and Paraffin Embedding (FFPE) to allow for extended storage To recover nucleic acids from an FFPE sample the paraffin must be disassociated from the sample. This is typically done by placing the sample in an organic solvent, such as xylene, to dissolve the paraffin, or by heating the sample in mineral oil. DNA sequencing applications require controlled fragmentation of the DNA as part of the library preparation workflow. Covaris AFA technology, the standard DNA fragmentation method for NGS applications, can be used for the direct fragmentation of DNA in the crude extract, thus reducing the number of downstream steps, simplifying the overall library preparation protocol and increasing yield. As in other AFA DNA shearing application, the DNA fragment size is controlled by the amount of energy delivered by the Focused- ultrasonicator, making it tunable to the specific size needed. • Samples processed in a 24 microTUBE rack – 96 plates are in development • Paraffin removal to DNA fragmentation without sample transfer • DNA purification compatible with robot friendly bead based methods • DNA ready for library preparation in ½ day Consistent DNA Extraction – High Yield & Quality – Uniform performances across sample input (Optional) – Integrated DNA Shearing High yield DNA extraction from 20 µm thick FFPE sections. Serially sectioned replicates were processed with the sFFPE DNA Extraction Kit with and without optional integrated DNA fragmentation step, and compared to the Promega® ReliaPrep™ FFPE gDNA Miniprep system. Uniform performance across large sample input range. DNA yield increased linearly with increasing FFPE section thickness from 10 to 40 µm, demonstrating the wide working range of the sFFPE DNA Extraction Kit. Extraction was done with the optional fragmentation step. Fragments size distributions of DNA extracted from FFPE block without the optional fragmentation step. Top: 10 scrolls processed as replicates from a well conserved kidney sample. Bottom: 10 scrolls processed as replicates from a different kidney sample. DNA fragment distribution profiles after direct fragmentation in the crude extract. Replicates fragmented at 300bp (top) or 200 bp (bottom). Samples were 20 µm thick sections from FFPE kidney blocks. NGS Microarray qPCR Analytical Readout FFPE Nucleic Acids Extraction Workflow Covaris FFPE Kit with AFA Protocol of Choice Paraffin Removal and Tissue Rehydratation Tissue Digestion Reverse Crosslinks DNA Fragmentation (optional) DNA Purification There are several drawbacks to these processes. • Not compatible with automation • Highly variable • Requires handling of toxic chemicals With Covaris Focused-ultrasonicators and FFPE kits, paraffin is removed from the samples by a purely mechanical process without the use of organic solvents. Paraffin is emulsified and sample rehydrated in less than 30 seconds AFA Process Starts 40 µm scroll smartPREP E-series • Circular transducer • 1 to 96 sample processing • Robot integration for full automation L-series • Linear transducer – Parallel processing • Highly uniform acoustic field along a row treats a full 96 plate 8x faster S-series • Circular transducer • Single sample processing M-series • Circular transducer • Single sample processing • Integrated temperature control Enabling smartPREP™ - Focused-ultrasonicators with AFA™ AFA Technology • Precisely matches the energy to the sample type • Yields reproducible results, sample-to-sample, day-to- day • Simplifies workflow and is easily automated • Provides end-to-end control of sample preparation HIGH HIGH Sample Mass LOW AFA Power Biomarkers Extraction Immunoassay Improvement Cell Lysis Drug Micronization FFPE Sample Nucleic Acids Extraction Controlled Crystallization Nanoliposome Formation Chromatin Shearing DNA Shearing Focused-ultrasonic Transducer Sample Vessel Focal Zone
Transcript of Improved workflow for FFPE tissue Nucleic Acid...
Guillaume DurinEdwin RuddGary SmejkalJohn-David HerlihyJim Laugharn Covaris, Inc. • 14 Gill Street, Unit H, Woburn, Massachusetts 01801-1721 USA
Tel: +1 781-932-3959 • Fax: +1 781-932-8705 • Email: [email protected]: www.covarisinc.com
Improved workflow for FFPE tissue Nucleic Acid extractionParaffin removal – Emulsification without organic solvent
Seamless Integration with NGS applications – High throughput capable
Tissue samples are commonly preserved by Formalin Fixation and Paraffin Embedding (FFPE) to allow for extended storage
To recover nucleic acids from an FFPE sample the paraffin must be disassociated from the sample. This is typically done by placing the sample in an organic solvent, such as xylene, to dissolve the paraffin, or by heating the sample in mineral oil.
DNA sequencing applications require
controlled fragmentation of the DNA as part of
the library preparation workflow. Covaris AFA
technology, the standard DNA fragmentation
method for NGS applications, can be used for
the direct fragmentation of DNA in the crude
extract, thus reducing the number of
downstream steps, simplifying the overall
library preparation protocol and increasing
yield. As in other AFA DNA shearing application,
the DNA fragment size is controlled by the
amount of energy delivered by the Focused-
ultrasonicator, making it tunable to the specific
size needed.
• Samples processed in a 24 microTUBE rack – 96 plates are in development
• Paraffin removal to DNA fragmentation without sample transfer
• DNA purification compatible with robot friendly bead based methods
• DNA ready for library preparation in ½ day
Consistent DNA Extraction – High Yield & Quality – Uniform performances across sample input
(Optional) – Integrated DNA Shearing
High yield DNA extraction from 20 µm thick FFPE sections. Serially sectioned replicates were processed with the sFFPE DNA Extraction Kit with and without optional integrated DNA fragmentation step, and compared to the Promega® ReliaPrep™ FFPE gDNA Miniprep system.
Uniform performance across large sample input range. DNA yield increased linearly with increasing FFPE section thickness from 10 to 40 µm, demonstrating the wide working range of the sFFPE DNA Extraction Kit. Extraction was done with the optional fragmentation step.
Fragments size distributions of DNA extracted from FFPE block without the optional fragmentation step. Top: 10 scrolls processed as replicates from a well conserved kidney sample. Bottom: 10 scrolls processed as replicates from a different kidney sample.
DNA fragment distribution profiles after direct fragmentation in the crude extract. Replicates fragmented at 300bp (top) or 200 bp (bottom). Samples were 20 µm thick sections from FFPE kidney blocks.
NGS
Microarray
qPCR
Analytical ReadoutFFPE Nucleic Acids Extraction Workflow
Covaris FFPE Kit with AFA Protocol of Choice
Paraffin Removal
and Tissue Rehydratation
Tissue Digestion
Reverse Crosslinks
DNA Fragmentation
(optional)DNA Purification
There are several drawbacks to these processes.
• Not compatible with automation
• Highly variable
• Requires handling of toxic chemicals
With Covaris Focused-ultrasonicators and FFPE kits, paraffin is removed from the samples by a purely mechanical process without the use of organic solvents. Paraffin is emulsified and sample
rehydrated in less than 30 secondsAFA Process Starts40 µm scroll
smartPREP E-series
• Circular transducer
• 1 to 96 sample processing
• Robot integration for full automation
L-series
• Linear transducer – Parallel processing
• Highly uniform acoustic field along a row treats a full 96 plate 8x faster
S-series
• Circular transducer
• Single sample processing
M-series
• Circular transducer
• Single sample processing
• Integrated temperature control
Enabling smartPREP™ - Focused-ultrasonicators with AFA™ AFA Technology
• Precisely matches the energy to the sample type
• Yields reproducible results, sample-to-sample, day-to-day
• Simplifies workflow and is easily automated
• Provides end-to-end control of sample preparation
HIGH
HIGH
Sample Mass
LOW AFA Power
Biomarkers Extraction
Immunoassay Improvement
Cell Lysis
Drug Micronization
FFPE Sample Nucleic Acids
ExtractionControlledCrystallization
NanoliposomeFormation
Chromatin Shearing
DNA Shearing
Focused-ultrasonic Transducer
Sample Vessel
Focal Zone
