Reliable Purification of Amplifiable DNA from FFPE Using ...
Transcript of Reliable Purification of Amplifiable DNA from FFPE Using ...
©2013 Promega Corporation.
Reliable Purification of Amplifiable DNA from FFPE Using the Maxwell® CSC Instrument
Doug Horejsh, Ph.D., Sr. Research Scientist - R&D
Outline
• Introduction to DNA purification from Formalin Fixed, Paraffin Embedded (FFPE) samples
• Challenges of qualifying the extracted DNA for downstream amplification
• Data demonstrating the successful and consistent purification of gDNA from breast, lung and colon FFPE samples.
Why Use FFPE Samples? Valuable Resource for Pro- & Retrospective Studies
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• Provides opportunity to correlate disease state and tissue morphology with genotype (DNA) and/or gene expression (RNA)
• Common method for archival of pathology tissue specimens • Very common with cancer samples
• Some FFPE tissue archives date back 50 years or more
• Valuable tool for retrospective studies of disease
• Opportunities for MDx and CDx applications
Preservation of Samples by Formalin Fixing and Paraffin Embedding
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Denatures protein, crosslinks with DNA
Fix tissue (formalin, neutral pH)
Dehydrate 1. Ethanol 2. Xylene (Toluene)
Paraffin embed (hot wax)
Sectioning and Storage 1. Microtome 2. Store at room temperature
Challenges
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• Formalin fixation crosslinks proteins and nucleic acids • Nucleic acids are partially degraded/fragmented • Degree of fragmentation varies depending on:
• Sample type and age • Fixation conditions
• Formalin fixation effects downstream applications
• Crosslinking makes nucleic acid inaccessible to amplification enzymes • Formalin residuals inhibit enzyme activity during extraction and PCR • Degradation/fragmentation inhibits amplification
• Deparaffinization often necessitates use of xylene or other organics • Hazardous
Current Research Offerings for FFPE DNA Extraction Manual & Automated Extraction Options Available
Manual Kit
• ReliaPrep™ FFPE gDNA Miniprep System
Automated Systems
• Maxwell® 16 Instrument & Kits
• Maxwell® 16 FFPE Tissue LEV DNA Purification Kit
• Maxwell® 16 FFPE Plus LEV DNA
Purification Kit
For Research Use Only. Not for Use in Diagnostic Procedures
Maxwell® Instruments and Chemistry Multiple Instruments to Match Your Needs
IVD Instruments: Maxwell® CSC Instrument
• Blood gDNA Purification Kit currently available • FFPE gDNA Purification Kit (coming soon, today’s discussion) • IVD, 21 CFR Part 11 compliant
• Intended for In Vitro Diagnostic Use. This product is currently
only available in US.
Research Instruments: Maxwell® 16 Instruments
• Multiple purification kits available for a variety of sample types • Blood, plasma, serum, buffy coat, buccal swabs, tissue,
FFPE tissue, CSF, amniotic fluid, nasal washes • Available with (AS3000) or without (AS2000) sample tracking
and UV light for decontamination
• Intended for Research Use Only. Not For Use In Diagnostic Procedures.
LEV Kits Maxwell® CSC and Maxwell® 16
Research Instruments
Maxwell® Instruments and Chemistry Easy-to-Use, Particle Mover Automation
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LEV Plunger
Cleared Lysate
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3 4 5 6 7 2 8
1 4 5 6 2 8 2 1 4 5 6
Mixing
Capture
Binding
Washing &
Elution Elution Tube
(300µl) Elution Tube (30-100µl)
SEV Kits Maxwell® 16 Research
Instruments only
Maxwell® Instruments and Chemistry Improved Consistency with Less Hands-on Time
• Process 1 to 16 samples in 30-60 minutes depending on sample/kit
• Bench top automation provides:
• Consistent results
• High yields of gDNA or RNA depending on sample type
• Sample tracking available
• Configured for large (SEV, 200-400µl) or small (LEV, 30-100µl) elution volumes
• Compact footprint conserves valuable bench space
For Research Use For In Vitro Diagnostic Use
Easy Pre-processing 1. Scrape sections 2. Proteinase K solution and Incubation Buffer 3. Incubate the sample at 70°C overnight 4. Lysis Buffer 5. Transfer to Maxwell® 16 Kit cartridge 6. Run automated extraction protocol
• Maxwell® 16 FFPE Tissue LEV DNA Purification Kit (optimal purity) • Maxwell® 16 FFPE Plus LEV DNA Purification Kit (optimal yield) • For Laboratory Use
Maxwell® 16 Kits for DNA Purification from FFPE Optimized for Different Results
New Development Efforts: Maxwell® CSC FFPE DNA Extraction Kit • Optimize purity AND yield • Designed under Design Control and manufactured under cGMP • For In Vitro Diagnostic Use
Purification from Various FFPE Tissues
Maxwell® CSC System Nucleic Acid Extraction for IVD Use
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This product is only available in the USA and is intended for in vitro diagnostic use.
Maxwell® CSC Instrument Clinical Sample Concentrator
• Manufactured under cGMP • Class I Exempt Medical Device (Clinical
Sample Concentrator ) • IQ / OQ available • Windows 7 Software: WiFi Capable /
Data Ready
For more information on the Maxwell® CSC Instrument, view the recorded webinar at: http://www.promega.com/resources/webinars/worldwide/archive/maxwell-csc-cgmp-compliant-automated-dna-extraction-from-blood/
Development of a Maxwell® CSC FFPE DNA Kit
Goal: Develop an IVD kit for use on the Maxwell® CSC Instrument
• What are the key metrics to measure success in DNA purification from FFPE samples?
• Purified DNA quantity and quality
• Once the metrics are identified, how will the purification chemistry be characterized and tested?
• Robust chemistry
• Development characterization
• Stringent product requirements
• Verified through extensive testing
Typical In-Line Measures of Yield and Quality Use to Predict Downstream Assay Success
• Factors that impact quality:
• Purification chemistry contaminants
• Cross-linked templates
• Fragmentation
• Concentration by:
• Absorbance
• Fluorescent dye
• Amplification (qPCR)
• Purity by:
• Absorbance ratio
• Internal positive control (IPC) to measure inhibition
• All are used to predict likelihood of success in downstream assays
Assess DNA Quantity/Quality by Absorbance Not Reliable for DNA Extracted from FFPE Samples
• Note the large peak at 225nm in the Chemistry B preparation
• Concentration and ratios are not useful at low absorbance levels
• Absorbance and amplifiability may correlate, but several other factors play a role
Chemistry A Chemistry B
Large peak at 225nm No peak at 225nm
gDNA Extraction from Matched Lung Tissue FFPE Slides
Assess DNA Quantity/Quality by Absorbance Not Reliable for DNA Extracted from FFPE Samples
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breast breast colon colon lung lung
Chem A Chem B Chem A Chem B Chem A Chem B
ng/
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Tissue/method
Quantitation by Absorbance
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breast colon lung
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Quantitation by Amplification
Chem A
Chem B
Large difference in quantitation • Absorbance at 260nm may not
be an accurate measure of amplifiable yield
Purity Ratios May Be More Predictive of Amplification Success
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breast breast colon colon lung lung
Chem A Chem B Chem A Chem B Chem A Chem B
Tissue/Method
Purity by Absorbance Ratios Dark: 260/280 Light: 260/230
• These panels show that higher A260/230 ratios may correlate with amplification success
• Contribution of purification chemistry
• Study absorbance ratio against amplifiability
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breast colon lung
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Quantitation by Amplification
Chem A
Chem B
No Correlation between A260/A230 and Amplifiability
• Input normalized to A260; Samples run using quantitative PCR
• Variety of chemistries tested across a single tissue type (breast) to include a range of A260/A230 ratios
• No correlation between A260/A230 and amplifiability • For QC, a more predictive test is needed
Is an Internal Positive Control (IPC) Assay a Better Alternative?
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Sample 1 Sample 2
A260/A230 Ratio
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Sample 1 Sample 2
Relative Amp for IPC
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Sample 1 Sample 2
Target Amplification (ng/µl)
• The purity measure, A260/A230 ratio, fails to correlate with amplification • The internal positive control assay shows relative amplification that tracks
with amplification of a target in the FFPE sample
• Eluate inhibition is tested using a commercial internal positive control assay • Amplification target is added with master mix and purification eluates are
compared with no inhibition controls (water)
Many Factors Influence Amplifiability Yield, Purity, Inhibitors and Fragmentation
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Sample 3 Sample 4
Relative Amp for IPC
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Sample 3 Sample 4
Target Amplification (ng/µl)
• These two samples have similar concentrations and purity ratios • The IPC assay fails to track with amplification success for some samples
• Another factor appears to be playing a role here – fragmentation of DNA • Yield, purity ratios, inhibition, fragmentation are all involved
Chemistry Choice Affects Average gDNA Fragment Size
Chemistry A
Chemistry B
Highly fragmented, Less amplifiable, higher A260
More intact, More amplifiable
bp
Am
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• Important to note that fragmentation occurs as part of fixation
• Highly fragmented gDNA is less amplifiable, as the peak is about 100bp
• The difference in area under the curve >300bp is the advantage of Chem A over Chem B
Fragment Size Impacts Amplifiability
Chemistry A
Chemistry B
≈ 200bp
200bp
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…
102 201
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Am
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Amplification of 100bp target is at best 50% with an average template fragment size of 200bp qPCR amplicon – 100bp Sanger seq – 250bp
Benefits of Automated FFPE DNA Extraction with the Maxwell® CSC DNA FFPE Kit
No xylene use equals improved safety
Easy to use system means fewer errors
Reduced hands-on time increases efficiency
High quality, less fragmented DNA improves downstream assays
= Reliable Amplification
Prototype Chemistry Identified Based on Initial Testing Shows Promise
The entire process was optimized to ensure and improve robustness
Samples • Adjacent slides from
multiple tissues • Relative comparison to
competitor chemistry
Method 1. Deparaffinization with
mineral oil 2. Lysis / Pro K treatment 3. Decrosslinking 4. RNase treatment 5. Purification on Maxwell® 6. Elution
Parameters Studied in Design Development (Evaluation of Robustness)
• Tissue input
• Lysis volume
• Pro K:
• Amount
• Temperature
• Lot variability
• De-crosslinking:
• Time
• Temperature
• Lysate carryover
• Cartridge components
• Elution volume
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0.25 curls 0.5 curls 1 curl 2 curls 3 curls
N = 4 per condition T-test p<0.01 for all r-squared = 0.979
Tissue Input: Number of Curls Scales with Recovery
• Direct correlation between amount of FFPE tissue input and amount of DNA recovered
• Additional starting material had no impact on qPCR inhibition as measured with an IPC (data not shown)
Elu
ate
DN
A C
on
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(n
g/µ
l)
Blue Dye Added to the Lysis/Pro K Solution to Improve pre-processing
Benefits of Dye Inclusion: Fewer pipetting steps Easier aqueous transfer Sample tracking into cartridge Potentially better performance
• Pipetted colorless aqueous layer from under mineral oil layer in prototype • Added inert blue dye to Lysis/Pro K step – no effect on chemistry
Dye included in final kit
Extraction Cartridges Pre-processed Samples with & without Dye
qPCR-based Quantitation
Transfer of Aqueous Phase
Decrosslinking Time & Temperature Assessment 4 Hours at 80°C Optimal for Breast Samples
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• 4 slides each condition, samples not pooled
• 80°C for 4 hours produces most amplifiable DNA for both 100bp and 300bp amplicons
100bp Amplicon (pg/µl)
300bp Amplicon (pg/µl)
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100bp Amplicon (pg/µl)
300bp Amplicon (pg/µl)
Decrosslinking Time & Temperature Assessment 4 Hours at 80°C Optimal for Colon Samples
• 4 slides each condition, samples not pooled
• 80°C for 4 hours produces most amplifiable DNA for both 100bp and 300bp amplicons
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• 4 slides each condition, samples not pooled
• 80°C for 4 hours appears to be better for the 100bp amplicon, with minor effect on 300bp amplicon
100bp Amplicon (pg/µl)
300bp Amplicon (pg/µl)
Minor decrease
Best
Decrosslinking Time & Temperature Assessment Minor Decrease for Lung Samples at 80°C, 4 Hour
Decrosslinking Experiments Summary 4 Hours at 80°C Provided Best Overall Results
• 4-hour decrosslinking at 80°C improved performance (amplification of larger amplicons) for most tissues
• Majority of samples tested
• 4-hour decrosslinking had minor impact on performance when 1-hour decrosslinking was optimal
• Minority of samples tested
4-hour decrosslinking is used in the final kit format
No Inhibition Observed Using Internal Positive Control (IPC) Assay
• Eluate inhibition is tested using a commercial internal positive control assay • Amplification target is added with master mix and purification eluates are
compared with no inhibition controls (water)
• No inhibition seen in these eluates using a common commercial qPCR master mix
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No inhib Breast Colon LungRe
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mp
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f IP
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Sample eluate (exp n=6, PCR n=2)
• Another check-in compared with competitor to complete development and lock chemistry/process for verification
Promega vs. Competitor FFPE DNA Extraction More Amplifiable DNA Extracted with Promega Kit
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QiaAmp FFPE DNA Promega FFPE DNA
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102bp Amplicon
Mounted Slides (5 micon): Breast Tumor Tissue, n=4
Competitor Promega
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QiaAmp FFPE DNA Promega FFPE DNA
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Competitor Promega
Maxwell® CSC FFPE DNA Purification Protocol ~5 Hours of Preprocessing + 50 Minute Run Time
Step Action
1 Scrape entire paraffin sample into individual tube.
Spin full speed briefly in microfuge to bring material down.
2 Add 300µl of mineral oil to each sample
Vortex 10 seconds to mix
3 Incubate at 80°C for 2 minutes
Vortex to mix
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Prepare a master mix of the Lysis buffer, Proteinase K and blue dye as shown below.
Reagent Amount/reaction Reactions (n+2) Total
Lysis Buffer 224µl 18 4032µl
Proteinase K 25µl 18 450µl
Blue Dye 1µl 18 18µl
5 Add 250µl of master mix to each sample tube, and vortex for 5 seconds.
6 Centrifuge at 10,000 x g for 20 seconds to separate layers. Gently mix aqueous phase with pipet to
break up pellet if present.
7 Incubate at 56°C for 30 min
8 Incubate at 80°C for 4 hours
9 Allow the sample to cool to room temperature. (~5 minutes)
10 Add 10µl of RNase A directly to the lysed sample in the lower phase. Pipet to mix
Incubate at room temp for 5 minutes
11 Spin sample full speed 2min. Add sample to cartridge, leaving pellet if present
12 Run Method
Similar Extraction Results from Ribbon Curl and Slide Samples
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Breast Colon Lung
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RNaseP
TERT 164
Ribbon Curls Slides
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Breast Colon Lung
RNaseP
TERT 164
No Increase in PCR Inhibitor Co-purification with Increasing Tissue Section Input
Group Number of samples
Mean Ct Mean Ct
No inhibition control 12 30.79 0.00
Double Tissue Input Samples 16 30.72 0.07
IPC Block 2 No Ct N/A
• Purifications done from either one or two sections to assess linearity • IPC assay was performed to ensure that no inhibitors were co-purified • Eluate DNA concentration is linear with input
Run Number of samples
Mean Ct Mean ΔCt
No inhibition control 12 30.73 0.00 Breast 16 30.63 0.11 Colon 16 30.71 0.02 Lung 16 30.66 0.08
IPC Block 2 No Ct N/A
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RNase P
TERT 164
Eluates are Stable Under Multiple Storage Conditions
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Breast Colon Lung
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Tissue
RNase P
TERT
• Eluates were stored for 1 week at 4°C or for 1 month at -20°C • 102bp and 164bp amplicons were tested
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Breast Colon Lung
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RNaseP
TERT 164
1 Week at 4°C 1 Month at -20°C
Summary
• Interest in FFPE samples in the molecular diagnostics lab is rapidly increasing
• Molecular and Companion Diagnostic assays
• Target amplification-based metrics are the most useful quality control
• Absorbance, absorbance ratios, and IPC can be affected
• Assessment by qPCR ensures downstream success in PCR-based assays
• Promega has developed a new FFPE DNA kit for use on the Maxwell® CSC Instrument
• Kit was developed for in vitro diagnostic use in combination with the Maxwell® CSC Instrument
• Intended for use with breast, lung and colon FFPE samples
• Well controlled manufacturing processes – manufactured under cGMP
• Consistently extracts more amplifiable DNA than competitor kits
©2013, Promega Corporation.
Thank you for your time and attention Questions are welcome