APPLICATION NOTE
Thermal cyclers
Thermal cycler sample
amplification uniformity: a
comparison of several models
Introduction
Thermal cycler well-to-well amplification uniformity
is a critical factor that can affect analysis of PCR
results. Without uniform reaction conditions across the
block, users cannot make robust conclusions when
comparing data obtained from wells at two different
locations. This study compares the reaction yield of
several thermal cyclers by measuring post-reaction
absolute fluorescence. Well-to-well amplification
uniformity is then compared by calculating the
coefficient of variance (CV) of fluorescence readings
obtained by each thermal cycler.
Materials and methods
The instruments tested in this study are shown in
Table 1. The same equipment, methods and reagents
were use for each instrument.
Table 1. Instruments tested for amplification uniformity.
Manufacturer
Model name
Cat. No.
Thermo Fisher Scientific
Applied Biosystems™ ProFlex™ 96-Well PCR System
4484075
Thermo Fisher Scientific
Applied Biosystems SimpliAmp Thermal Cycler
A24811
Bioer
GeneMax™ Thermal Cycler
BYQ6067
Bio-Rad
C1000 Touch Thermal Cycler with 96-Well Fast Reaction Module
185-1196
Bio-Rad
T100™ Thermal Cycler
186-1096
Eppendorf
™
Mastercycler Nexus Gradient
6331 000.017
Eppendorf
Mastercycler™ Nexus GX2
6336 000.015
Eppendorf
Mastercycler Pro S
6325 000.510
SensoQuest
Labcycler Gradient
011-101, 012-103
Takara
Dice Touch
TP350
™
™
™
™
™
Sample preparation
A single bulk reaction was prepared using
Applied Biosystems™ AmpliTaq Gold™ 360
Master Mix (Cat. No. 4398886) according to the
standard protocol. Invitrogen™ Lambda DNA
Standard (component C) (Cat.No. P7589) was
used as the template at a final concentration
of 0.01 ng/µL with forward primer
(5´-GATGAGTTCGTGTCCGTACAACT-3´) and reverse
primer (5´-ACGGCTGCACGGAGTTCAGTATG-3´) at
0.2 µM. The bulk reaction was divided between nonoptical 8-tube strips and non-optical 96-well plates
(not created by the instrument manufacturers) at 10 µL
or 100 µL testing volume. The following thermal profile
was used for all instruments in this study: a primary
stage at 94°C for 10 minutes, a secondary stage of
25 cycles at 94°C for 15 seconds and 70°C for 90
seconds, and a final stage at 72°C for 7 minutes.
Data acquisition and analysis
After completion of thermal cycling, Applied
Biosystems™ Power SYBR™ Green PCR Master Mix
(Cat. No. 4368577) was added to each reaction and
incubated for 10 minutes. The reactions were then
transferred to an Applied Biosystems™ MicroAmp™
Optical 96-Well Reaction Plate (Cat. No. N8010560)
for fluorometric analysis with the Applied Biosystems™
ViiA™ 7 Real-Time PCR System (Cat. No. 4453534).
Amplification uniformity was calculated by %CV as
stated in the following formula:
%CV =
Standard deviation of SYBR Green signal over 3 runs
Average SYBR Green signal over 3 runs
x 100%
Results
Figure 1 shows the average fluorescence intensity
obtained from each thermal cycler over two reaction
volumes and two plastic consumable types. The
individual values are specified below the chart, and
the group average of 7,253,062 fluorescence units is
indicated as a red line. Figure 2 shows the %CV for
each plastic consumable and reaction volume. The
%CV group average of 10.3% is shown as a red line.
Average fluorescence intensity
14,000,000
Fluorescence intensity
12,000,000
10,000,000
8,000,000
7,253,062
6,000,000
4,000,000
2,000,000
0
ProFlex 96-Well
SimpliAmp
PCR System
Thermal Cycler
GeneMax
Thermal Cycler
C1000 Touch
Thermal Cycler
T100
Thermal Cycler
Mastercycler
Nexus GX2
Mastercycler
Nexus Gradient
Mastercycler
Pro S
Labcycler
Gradient
Dice
Touch
10 μL, 96-well plate
8,051,637
7,910,946
9,239,160
7,946,722
8,504,935
ND
8,819,051
8,920,168
5,550,320
8,055,912
10 μL, 8-tube strips
7,121,495
7,367,679
8,041,636
7,175,663
7,869,934
7,815,814
7,170,831
8,212,778
6,656,877
7,514,640
100 μL, 96-well plate
9,581,911
10,207,855
7,977,774
8,614,839
5,016,273
ND
3,116,432
6,761,303
2,931,623
3,624,982
100 μL, 8-tube strips 10,470,426
11,303,278
9,375,210
9,690,691
6,355,783
3,785,679
3,273,543
8,073,197
3,265,825
4,243,518
Instrument
Figure 1. Average fluorescence intensity obtained from thermal cyclers.
ND = not determined.
Uniformity measured by %CV
25.0
20.0
%CV
15.0
10.3
10.0
5.0
0.0
ProFlex 96-Well
SimpliAmp
PCR System
Thermal Cycler
GeneMax
Thermal Cycler
C1000 Touch
Thermal Cycler
T100
Thermal Cycler
Mastercycler
Nexus GX2
Mastercycler
Nexus Gradient
Mastercycler
Pro S
Labcycler
Gradient
Dice
Touch
22.0
10 μL, 96-well plate
5.8
5.2
5.1
10.6
14.9
ND
7.0
6.7
11.1
10 μL, 8-tube strips
7.3
9.0
11.0
5.8
12.2
4.9
6.3
6.8
6.3
9.7
100 μL, 96-well plate
7.3
7.3
12.7
7.5
15.0
ND
8.6
14.7
6.7
21.6
100 μL, 8-tube strips
9.6
8.0
15.6
6.0
20.0
13.1
10.5
12.6
7.2
20.8
Instrument
Figure 2. Average %CV of thermal cyclers.
ND = not determined.
Discussion
The ability of a thermal cycler to uniformly amplify
each well of the reaction plate is a pivotal and
often overlooked requirement for the user when
designing experiments. In this study, we have shown
that PCR amplification uniformity varies between
thermal cyclers. This was demonstrated as a
side-by-side comparison while utilizing the same
reaction chemistry.
For best results, we recommend using a thermal
cycler with both high reaction yield and good wellto-well uniformity (as indicated by low %CV). The
combined results for each volume and plastic
consumable suggest that the Applied Biosystems™
brand of thermal cyclers meet these criteria.
Find out more at thermofisher.com/thermalcyclers
For Research Use Only. Not for use in diagnostic procedures. © 2015 Thermo Fisher Scientific Inc. All rights reserved. All trademarks
are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. AmpliTaq Gold is a trademark of Roche Molecular
Systems, Inc. C1000 Touch and T100 are trademarks of Bio-Rad Laboratories, Inc. Mastercycler is a trademark of Eppendorf AG. Dice is a
trademark of Takara Bio Inc. GeneMax is a trademark of Bioer. CO210556 0715