Evaluation of Maxwell 16 for Automated Nucleic Acid
Extraction from Whole Blood and Formalin-Fixed
Paraffin Embedded (FFPE) Tissue
Khokhar,
1
SK ,
Leos,
1
NK ,
Mitui,
1
M,
Park,
1,2
JY and
Rogers,
3,4
BB
Departments of Pathology, 1Children’s Medical Center Dallas, 2 The University of Texas Southwestern
Medical Center, Dallas, TX ; 3Children’s Healthcare of Atlanta and ⁴Emory University, Atlanta, GA
Results
FFPE tissue DNA and RNA extraction
Mean FFPE RNA concentration
Nucleic acid extraction is one of the most technically demanding and
labor intensive procedures performed in molecular diagnostic
laboratories. Manual sample preparation methods are time
consuming and susceptible to contamination, handling errors and
variation. Automated nucleic acid extractors are successful in
extracting clinical specimens due to their efficient recovery, lack of
cross contamination and ease of performance. As a new method or
technology becomes available, it is ideal to assess and compare it
with the existing methods before being applied to clinical use. The
objective of this study was to evaluate the performance of Maxwell
16, (Promega, Madison, WI) in extracting nucleic acid from different
specimen types and to compare it to a manual (Qiagen, Valencia, CA)
and an automated nucleic acid extraction (Biomerieux, Durham, NC)
methods.
Sa Maxwell
Automated
Sa prep kit)
Manual (All
Sa
FFPE sections
Sa
FFPE sections
Sa Maxwell
Automated
FFPE sections
Sa
Deparaffinize
Sa and lyse
Digest
Sa
Digest
Sa
Cool and centrifuge to separate RNA in
Sa
the supernatant and DNA in the pellet
Lyse
Sa
Concentration (ng/µL)
Materials and Methods
Background
Lyse
Sa
Lyse pellet
Sa
Incubate RNA
Sa
supernatant at 80°C
Apply to QIAamp spin
column to bind
Sa genomic
DNA
Add ethanol
Sa
Collect
Sa RNA
Wash
Sa
Elute
SaDNA
DNAse
Satreat
DNase
Satreat
Wash
Sa
Load cartridge
Sa
Lyse
Sa
Bind
Sa
Wash
Sa
60
40
Qiagen
6 7
8
9 10
1 2
3
4 5
6 7
8
9 10
1 2
3
4 5
6 7 8
9 10
NEC
NAC
4 5
NEC
100
3
627 bp
80
60
40
NAC
0
NEC
627 bp
20
Figure 3: Quantity and quality assessment of extracted DNA from whole blood samples A) DNA concentration obtained by Maxwell was
significantly greater than either EasyMag (p<*0.0001) or QiaAmp Blood DNA kit, Qiagen (p<*0.001), n=10, for all methods. Mean DNA purity
(260/280) ratio was 1.84, 1.58 and 1.84 for Maxwell, EasyMag and QiaAmp Blood DNA kit, respectively. B) β-actin PCR and Agarose gel
electrophoresis demonstrate the amplification product of the extracted DNA from all methods. The numbers 1-10 represent 10 different whole
blood DNA extracts from three different methods.
200
160
120
80
*p<0.0001
Maxwell
2
7 14 19
2
7 14 19
NEC
NAC
Mean FFPE DNA concentration
Qiagen
NEC
B
Maxwell
Maxwell FFPE RNA had no detectable GAPDH in all samples
All prep FFPE DNA/RNA kit (Qiagen) had detectable GAPDH in 9 of 19 samples
Of the 9 positive Qiagen FFPE RNA extracts, 5 amplified between 31-35 threshold
cycles and 4 amplified between 36-39 threshold cycles
NOTE: Maxwell, did not have a specific FFPE RNA kit at the time of this study. This
protocol was designed using the Tissue LEV kit from Promega, along with reagents
from the FFPE DNA kit
Conclusions
Qiagen
NEC
120
A
Off –board
Sa Lysis
Silica
Sa
EasyMag
1 2
DNA Ladder
Sa
Manual QiaAmp
kit
Maxwell
140
concentration (ng/µL)
Off –board
Sa Lysis
Automated
Sa EasyMag
*p<0.001
EasyMag
NEC
Concentration (ng/µL)
*p<0.0001
B
Maxwell
NEC
Mean Whole Blood DNA concentration
A
DNA Ladder
Results
Whole Blood DNA extraction
Automated
Sa Maxwell
80
Figure 5: Quantitative assessment of extracted RNA from FFPE. No significant
difference in the FFPE RNA yield between Maxwell (Promega) and AllPrep FFPE
DNA/RNA Kit (Qiagen) was noted (p=0.098), n=19 for both methods. FFPE Mean
RNA purity (260/280 ratio) was 1.4 and 2.2 for Maxwell and Qiagen, respectively.
Apply to RNeasy spin
Sa
column
Collect
Sa DNA
Materials and Methods
Sample
Sa
Qiagen
0
Figure 2: Schematic representation of the two different extraction methods employing Maxwell (Promega) and AllPrep DNA/RNA FFPE kit
(Qiagen) for extracting DNA/RNA from FFPE tissue. The starting material for all these methods was two 20µM section of FFPE liver tissue.
To assess the quantity and purity of extracted nucleic acid from whole
blood and formalin-fixed paraffin embedded tissue (FFPE).
To compare the ability of these methods to yield high quality
extracted nucleic acid as assessed by PCR testing.
100
20
Elute
SaRNA
Specific Aims
Maxwell
120
Qiagen
286 bp
Maxwell 16, Promega efficiently extracts DNA from whole blood, is semiautomated and allows for relatively high throughput processing of samples
Whole Blood DNA : Maxwell performs better than either EasyMag or QiaAmp
Blood DNA Kit in terms of quantity of DNA extracted. All three extraction methods
(Maxwell 16, EasyMag and QiaAmp Blood DNA kit) performed well in terms of
amplification and purity
FFPE DNA: Manual method using AllPrep FFPE DNA/RNA kit performs better than
Maxwell. However, unsuccessful PCR amplification of 2/4 representative extracts
from both methods suggest DNA degradation or inhibitors
FFPE RNA: Both AllPrep FFPE DNA/RNA kit and Maxwell perform similarly in
terms of quantity. However, the Maxwell FFPE RNA extraction method requires
further optimization due to unsuccessful amplification of RNA extracts
Future studies
Optimization of the FFPE Maxwell and manual RNA extraction methods
Expand assessment to other specimens (e.g., fresh-frozen tissue)
References
AllPrep DNA/RNA FFPE Handbook (80234) and QiaAmp DNA Blood Mini Kit
Handbook (51104), Qiagen
Maxwell 16 LEV Blood DNA Kit technical manual (TM333), FFPE Tissue LEV DNA
Purification Kit technical manual (TB382) and Maxwell 16 Tissue LEV Total RNA
Purification Kit technical manual (TB367), Promega
C
40
0
Collect
Sa
Collect
Sa
Elute
Sa
Figure 1: Schematic representation of three different extraction methods employing Maxwell 16
(Promega), EasyMag (Biomerieux) and QiaAmp Blood DNA Kit (Qiagen ) for extracting DNA from whole
blood sample. All these methods are silica based.
Quality score: 42
Quality score: 34
Figure 4: Quantity and quality assessment of extracted DNA from FFPE tissues. A) FFPE DNA concentration obtained by AllPrep FFPE DNA/RNA
Kit, Qiagen was significantly greater than that by Maxwell (p<*0.0001). FFPE Mean DNA purity (260/280 ratio) was 1.9 and 2.0 for Maxwell and
Qiagen, respectively, n=19 for both methods. B) CDKL5 PCR and Agarose gel electrophoresis for exon 3 show amplification of 2/4 extracts from
both methods. C) CDKL5 exon 3 Sequencing of the PCR product shown in Figure 4B demonstrates that the extracted DNA from both methods
could be used for downstream applications.
Acknowledgements
We thank Michael Gallegos, BA and Dania Lopez, MT for technical assistance, Matt
Quintero, PhD for editorial review, and Rong Huong for statistical analysis.