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Promega Corporation Simple and Reliable Automated Purification of High Quality Nucleic Acids from Blood Charles Cowles, Ph.D. Sr. Research Scientist – Promega R&D
Promega Corporation
Simple and Reliable Automated Purification of High Quality
Nucleic Acids from Blood
Charles Cowles, Ph.D.
Sr. Research Scientist – Promega R&D
Promega Corporation 2
Webinar Outline
• Why extract nucleic acids from blood?
• Challenges in extracting nucleic acids from blood
• Introduction to the Maxwell® CSC Instrument and Kits
• Maxwell® CSC Blood DNA Kit
• Protocol
• Features
• Performance
• Maxwell® CSC Blood RNA Kit
• Protocol
• Features
• Performance
Promega Corporation 3
Why Extract Nucleic Acids from Blood?
Blood samples are:
• Cheap
• Far less invasive than tissue biopsies
• Easily obtained in an outpatient clinic
Nucleic Acids from blood can provide:
• Targeted and shotgun sequence
information for pharmacogenomics,
mutation analysis, and genotyping
• Qualitative information to enable
disease diagnosis and ongoing
monitoring of status
• Quantitative information to aid in
disease therapy
Promega Corporation 4
Challenges in Extracting Nucleic Acids from Blood
Blood is:
• Extremely high in protein content – difficult to extract clean
nucleic acids
• Far less dense in cell count than solid tissue – yield is less
from similar starting volumes
• Relatively low in nucleic acid content (only WBCs have a
nucleus, yet they comprise <1% of the total cellular content of
blood) – again, lower yields than similar tissue volumes
• High in extracellular RNases – increases risk of RNA
degradation during extraction
Promega Corporation 5
Maxwell® CSC Instrument Clinical Sample Concentrator for In Vitro Diagnostic Use
• A Class I Exempt Medical Device in
the U.S. and Canada
• Manufactured under cGMP
• Tablet-driven with Windows 7
Software and is data-ready
• Supported with Installation and
Operational Qualification (IQ/OQ)
Services
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/
Promega Corporation 6
Maxwell® CSC System Purification Kits Available for Key Sample Types
This product is only available in the USA and Canada and is intended for in vitro diagnostic use.
• Blood DNA Kit
• Blood RNA Kit – recently launched
• FFPE DNA Kit
• FFPE RNA Kit
Promega Corporation 7
Maxwell® CSC Instrument Easy-to-Use, Particle Mover Automation
1 3 4 5 6 7 2
LEV Plunger
Add Sample
1 3 4 5 6 7 8
3 4 5 6 7 2 8
1 4 5 6 2 8
Mixing
Capture
Binding
Washing &
Elution Elution Tube (30-100µl)
Parallel processing of 1-16 samples at a time
Promega Corporation 8
Benefits of Automating Nucleic Acid Extraction from Blood Using the Maxwell® CSC System
Ease of use
Reduced hands-on time for increased efficiency and throughput
Improved consistency of extractions vs. manual kits
Minimal downstream PCR inhibition due to low levels of contaminants
in extracted nucleic acids
Optimized DNA and RNA yield across a wide range of white blood cell
(WBC) counts
= Reliable Amplification
Promega Corporation 9
Webinar Outline
• Introduction to the Maxwell® CSC Instrument and Kits
• Why extract nucleic acids from blood?
• Challenges in extracting nucleic acids from blood
• Maxwell® CSC Blood DNA Kit
• Protocol
• Features
• Performance
• Maxwell® CSC Blood RNA Kit
• Protocol
• Features
• Performance
Promega Corporation 10
Incubate 20 min. at 56oC
Vortex lysate
50 – 300 μl Whole Blood
Run-time = 38 minutes
Proteinase K and Lysis Buffer
+
Load Cartridge
Maxwell® CSC Blood DNA Extraction Protocol Quick, 1 Hour Process
Promega Corporation 11
• High gDNA quantity & quality
• Broad collection tube compatibility
o Heparin, EDTA or sodium citrate
• Flexible blood storage conditions
• Highly linear yield over a wide range of WBC inputs
• Consistent gDNA yield and quality over time
• Consistent yield and quality from instrument-to-instrument
Maxwell® CSC Blood DNA Kit Features
Promega Corporation 12
Performance Requirements Based on Your Needs
Property Requirement Typical Performance
gDNA Concentration ≥80ng/µl 150 - 400ng/µl
Purity (A260/A280) >1.7 1.85 - 1.95
Purity (A260/A230) >1.5 1.9 - 2.2
Based on:
• 300 ml whole blood per extraction
• 4 x 106 – 10 x 106 white blood cells/ml
• 50 ml elution volume
Promega Corporation 13
Broad Collection Tube Compatibility Works with EDTA, Heparin, or Sodium Citrate Tubes
0
1
2
3
4
5
6
EDTA Heparin Sodium Citrate
DN
A Y
ield
(µ
g/1
06 W
BC
)
Anticoagulant Tube Type
0
0.5
1
1.5
2
2.5
EDTA Heparin Sodium Citrate
Ab
so
rba
nc
e R
ati
o
Anticoagulant Tube Type
A260/A280 A260/A230
* n=8
Consistent Performance Regardless of Anticoagulant
Collection Tube Used
A260/A230 A260/A280
Promega Corporation 14
0
1
2
3
4
5
6
Ambienttemperature for
3 days
4°C for 7 days -80°C thenthawed
DN
A Y
ield
(µ
g/1
06 W
BC
)
Storage Conditions
0
0.5
1
1.5
2
2.5
Ambienttemperaturefor 3 days
4°C for 7days
-80°C thenthawed
Ab
so
rba
nc
e R
ati
o
Storage Conditions
A260/280 A260/230A260/A280 A260/A230
Robust Yield and Purity After Storing Samples under
Common Conditions
* n=8
Flexible Blood Sample Storage Equivalent Performance with Different Storage Conditions
Promega Corporation 15
Linear DNA Yield from Variable Input Volume Provides Flexibility to Adjust Input Volume
R² = 0.9969
0
2
4
6
8
10
12
14
0 50 100 150 200 250 300 350
DN
A Y
ield
(µ
g)
Input Blood Volume (µl)
* n=8
Promega Corporation 16
Low Day-to-Day Variability Consistent, 10-12 µg Yields Across Multiple Days
0
2
4
6
8
10
12
14
1 2 3 4 5
DN
A Y
ield
(m
g)
Consecutive Days
0.0
0.5
1.0
1.5
2.0
1 2 3 4 5
Ab
so
rban
ce R
ati
o
Consecutive Days
A260/A280 A260/A230A260/A280 A260/A230
* n=8
Promega Corporation 17
Low Instrument-to-Instrument Variability Consistent, 10-12 µg Yields Across Instruments
0
2
4
6
8
10
12
14
1 2 3
DN
A Y
ield
(µ
g)
Instrument Number
0.00
0.50
1.00
1.50
2.00
1 2 3
Ab
so
rban
ce R
ati
o
Instrument Number
A260/A280 A260/A230A260/A280 A260/A230
* n=8
Promega Corporation 18
Maxwell® CSC Blood DNA Kit Summary
Features of the Maxwell® CSC Blood DNA Kit:
• High gDNA Quantity & Quality
• Broad anti-coagulant tube compatibility
• Flexible blood storage conditions
• Protocol and chemistry optimized for a wide range of WBC counts
• Linear yield over a wide range of input volumes
• Reproducible gDNA yield and quality over time
• Consistent yield and quality from instrument-to-instrument
• Developed under Design Control and manufactured under cGMP
• Developed for in vitro diagnostic use in combination with the
Maxwell® CSC Instrument
Promega Corporation 19
Webinar Outline
• Introduction to the Maxwell® CSC Instrument and Kits
• Why extract nucleic acids from blood?
• Challenges in extracting nucleic acids from blood
• Maxwell® CSC Blood DNA Kit
• Protocol
• Features
• Performance
• Maxwell® CSC Blood RNA Kit
• Protocol
• Features
• Performance
Promega Corporation 20
Maxwell® CSC Blood RNA Purification Protocol ~30 Min. Preprocessing + ~1 Hour Instrument Run
Step Action
1 Pipette 2.5 mL of fresh, whole, EDTA-drawn blood into a conical tube.
2 Add 7.5 mL of Solution A. Invert 10 times to mix.
3 Incubate at room temperature for 10 minutes, repeat inversion twice in
this interval.
4 Centrifuge at 3,000 x g for 10 minutes to pellet the WBCs; Remove
supernatant.
5
Resuspend WBC pellet in Solution B (200 µl/sample) by vortexing. Add
Proteinase K (25 µl/sample) and Lysis Buffer (200 µl/sample), vortex to
mix.
6 Incubate homogenate at room temperature for 10 minutes.
Alternatively, homogenate can be frozen at -20◦C for up to 5 days.
7 Add DNaseI to cartridges. Load cartridges with homogenates.
Promega Corporation 21
Maxwell® CSC Blood RNA Kit Performance Requirements Based on Your Needs
• What are the key metrics of success in RNA purification from blood
samples?
Functional with input samples having a wide range WBC counts
High RNA quantity and quality, as measured by absorbance
Absence of significant RT-qPCR inhibitors in RNA eluates
Low levels of DNA contamination, as measured by qPCR
In-cartridge DNase I treatment
Stopping point built into protocol
Promega Corporation 22
Blood Volume and WBC Count Intended Use Range
• The Maxwell® CSC Blood RNA Kit was developed to process 2.5 mL
of whole, EDTA-drawn blood.
• The Maxwell® CSC Blood RNA Kit is intended to be used with
samples which have a WBC count of 4 x 106 to 10 x 106 WBCs / mL.
Promega Corporation 23
RNA Quantity and Quality Requirements by Absorbance and Typical Performance
Attribute Product
Requirement
Verification
Result
RNA Quantity ≥ 50 ng/μl 152.16 ng/μl
RNA Quality, A260/A280 ≥ 1.8 2.14
RNA Quality, A260/A230 1.8 < x < 2.4 2.116
n=8 replicates
Promega Corporation 24
RNA Quantity and Quality Requirements by RT-qPCR and Typical Performance
Attribute Product
Requirement
Verification
Result
RNA Quantity > 20 pg/μl 158.71 ng/μl
RNA Quality, IPC Cq < 2 cycles 0.090 cycles
n=8 replicates
Promega Corporation 25
DNA Contamination by Absorbance and qPCR and Typical Performance
Sample ID Yield (ng/μl) gDNA
(copies/μl) % gDNA
1 145.19 125 0.28
2 163.77 369 0.74
3 138.63 23 0.05
4 159.43 43 0.09
5 159.77 35 0.07
6 146.65 320 0.72
7 154.36 56 0.12
8 157.11 79 0.17
Average 153.11 131 0.28
Product requirement: Extracted RNA contains < 1% gDNA
• Measured total nucleic acid by absorbance (ng/μl) and gDNA by qPCR (copies/μl)
• Related these values assuming 1 human genome copy = 3.3 pg DNA.
Promega Corporation 26
Built-In Pre-processing Protocol Stopping Point Lysates Can Be Stored Frozen Before Running Extraction
Attribute Product
Requirement
Verification
Result
RNA Quantity, Absorbance ≥ 50 ng/μl 159.01 ng/μl
RNA Quantity, RT-qPCR > 20 pg/μl 228.73 ng/μl
RNA Quality, A260/A280 ≥ 1.8 2.137
RNA Quality, A260/A230 1.8 < x < 2.4 2.133
Product requirement: The protocol should be flexible and should allow
partially processed samples to be frozen for ≥ 5 days without negatively
impacting RNA quantity, quality, or amplification.
n=8 samples were partially processed. The homogenates were
frozen for 6 days, then thawed and purification was completed.
Promega Corporation 27
Summary
Features of the Maxwell® CSC Blood RNA Kit:
• Protocol and chemistry optimized for a wide range of WBC counts
• Extracted RNA is of high quantity and quality and is suitable for
downstream RT-qPCR applications
• In-cartridge, hands-free DNaseI treatment
• Minimal contaminating gDNA or RT-qPCR inhibitors
• Optional stopping point built into pre-processing protocol
• Developed for in vitro diagnostic use in combination with the
Maxwell® CSC Instrument
• Developed under Design Control and manufactured under cGMP
Promega Corporation
Thank You for Your Time and Attention
Questions are
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