PRODUCT BULLETIN
TaqMan multiplex real-time PCR
Get more data out of your sample
• A complete multiplex real-time PCR
(qPCR) solution for gene expression
and genotyping applications
• Applied Biosystems™ ABY™ and
JUN™ dyes, QSY™ quencher, and a
multiplex master mix for optimal
amplification performance
• Up to 4-plex reactions—as sensitive
as singleplex reactions, decreases
the starting material required, and
minimizes optimization processes
Obtaining the maximum amount of
genetic information from an important
but small amount of sample can be
challenging. This is particularly true
with formalin-fixed, paraffin-embedded
(FFPE) samples or tumor biopsies that
are used for translational research
studies. Singleplex qPCR is frequently
used for these clinical research
samples, but this typically has a
higher cost per sample than running
in multiplex format. The additional
time and materials required to set up
multiple single-assay reactions could
also significantly increase the cost of a
complex project.
Multiplex qPCR, a strategy where
more than one target in a sample is
amplified and quantified in a single
tube, can decrease the quantity
of sample material and reagents
required. A complete solution for
multiplex qPCR is presented here,
TaqMan multiplex real-time PCR
Filters
wavelength
(nm)
Emission
spectra
~517 nm ~551 nm ~580 nm ~617 nm~654 nm
Dyes FAM VIC ABY JUN Mustang Purple
Figure 1. Fluorescence emission spectra of FAM, VIC, ABY, and JUN dyes used for multiplex
real-time PCR. Grey zones represent the filters available on Applied Biosystems™ real-time PCR
systems: 1 through 6 for the QuantStudio™ 7 or 12K Flex Real-Time PCR Systems; 1 through 5 for
the QuantStudio™ 6 Flex Real-Time PCR System, ViiA™ 7 Real-Time PCR System, and 7500 or 7500
Fast Real-Time PCR System. MP = Mustang Purple™ dye.
with components designed to work • The Applied Biosystems™
together for better data quality and TaqMan® Multiplex Master Mix was
less time for optimization. The solution developed to allow amplification of
consists of the following: 4 targets simultaneously, without
competition between targets. This
• Applied Biosystems™ TaqMan®
master mix contains the Applied
probes using QSY quencher,
Biosystems™ Mustang Purple™
providing maximal PCR efficiency in
dye, a passive reference used for
a multiplex format. These probes can
normalization instead of the Applied
be ordered with Applied Biosystems™
Biosystems™ ROX™ dye, allowing
FAM™ and VIC™ dyes and also with
for measurement of JUN dye in the
the ABY and JUN dyes, allowing
channel previously used to measure
amplification of up to 4 targets in a
ROX dye.
single reaction. These reporter dyes
are optimized to work together with
minimal spectral overlap for improved
performance (Figure 1). In addition,
the QSY quencher is fully compatible
with probes that have minor-groove
binder (MGB) quenchers.
• Off-the-shelf, predesigned assays— A
1.00 40.00
0.450
0.90

an RNase P assay using an ABY-

0.400
35.00
0.80
0.350

QSY probe and a GAPDH assay 0.70

30.00 0.300

Ct (std. dev.)
0.60

Ct (std. dev.)

Ct (average)
0.250
using a JUN-QSY probe. Both 0.50 25.00
0.200
0.40
assays are available in limited and 0.30
20.00
0.150

0.100
nonlimited primer concentrations. 0.20
15.00 0.050
0.10
0.000

• Calibration plates for ABY, JUN, and

0.00 10.00 2
200,000 20,000 2,000 200 20
Template per reaction (pg)
Mustang Purple dyes, available 1.00 40.00

B
1.00 40.00 0.450 40.00
in 96-well, 96-well Fast, and 384- 0.90
0.90
0.450
0.600
0.400
0.400
40.0040.00
0.200
35.00 0.500
0.80 35.00 35.00
well formats. 35.00 0.180
0.80 0.350 35.00
0.350 0.160
0.70 0.400 30.00
0.70 30.00

Ct (std. dev.)
0.300

Ct (average)
30.00 0.300 0.140

Ct (average)
Ct (std. dev.)
0.60 30.00

t t (std. dev.)

Ct (average)

Ct (std. dev.)
Ct (average)
Ct (std. dev.)
• Additional services provided through
0.60 0.300 30.0025.00

(std. dev.)

Ct (average)
0.250 0.120
0.250
0.50 25.00 0.100
0.50 25.00 0.200
0.200
0.200 20.00

our custom services program— 0.40 25.00

25.00
0.080

CC
0.40 0.150
20.00 0.150
0.100 15.00 0.060
0.30 20.00
0.30
save time and let our Applied 0.20
0.20 15.00
0.100
0.100
0.000
20.00
20.0010.00
0.040
0.020
15.00 0.050 200,000 20,000 2,000 200 20
0.050
Biosystems™ TaqMan® Assay 0.10
0.10
0.000
0.000
Template per reaction (pg) 15.00
15.00
0.000

0.00 10.00 200,000 2,000 200 20

0.00 10.00 20,000
experts design your 200,000
200,000
20,000
20,000
2,000
2,000
Templateper
perreaction
200
200
reaction(pg)
(pg)
20
20
200,000 20,000 2,000
Templateper
Template perreaction
200
reaction(pg)
(pg)
20

Template
multiplex assays.
1.00 40.00
C
0.600 40.00 0.450
0.600 40.00
0.200
0.90
0.200 28.00
28.00 0.400
0.500 35.00
This multiplex solution is compatible
0.500 35.00 0.180 35.00
26.00
0.180
0.80 26.00
0.160 0.350
0.400 30.00 0.160 24.00
24.00
0.400 30.00 0.70
dev.)

Ct (average)
with the Applied Biosystems™
(std.dev.)

Ct (average)
0.140 30.00 0.300
0.140 22.00
22.00

Ct (std. dev.)
Ct (std. dev.)

(average)
0.60

C (std. dev.)

Ctt (average)
0.300 25.00

Ct t(std. dev.)

Ct (average)
0.300 25.00 0.120
CCt (std.

0.120 20.00 0.250

20.00
QuantStudio™ 6, 7, and 12K Flex 0.50
0.100
0.100
25.00
t

0.200
0.200 20.00 18.00 0.200
20.00 0.080 18.00
0.40
0.080

C
Real-Time PCR Systems, as well as
16.00
16.00 0.150
0.100 15.00 0.060 20.00
0.100 15.00 0.060
0.30
14.00
14.00 0.100
0.040
0.040
the Applied Biosystems™ ViiA™ 7 Real- 0.000
0.000
200,000
200,000 20,000
20,000 2,000
2,000 200
200 20
20
10.00
10.00
0.20
0.020
0.020
0.10
12.00
15.00
12.00 0.050
0.000 10.00
10.00
0.000
Time PCR System and the Applied Templateper
Template perreaction
reaction(pg)
(pg)
0.00 200,000
200,000 20,000
20,000 2,000
2,000 200
200 2020 10.00
0.000
2
200,000 20,000Template
2,000
perreaction 200
reaction(pg)
(pg) 20
Template per
Biosystems™ 7500 and 7500 Fast D
Template per reaction (pg)

Real-Time PCR Systems. 0.600 40.00

0.200
0.500 35.00 0.180

Multiplexing without compromise

0.160
0.400 30.00
Ct (std. dev.)

Ct (average)
0.140

Ct (std. dev.)
0.300
The multiplex format enables cost
25.00 0.120
0.100
0.200 20.00

savings and preservation of limited 0.100 15.00

0.080
0.060

sample, but it’s important to obtain the 0.000 10.00

0.040
0.020
200,000 20,000 2,000 200 20
same sensitivity as in the singleplex Template per reaction (pg)
0.000

format. Figure 2 demonstrates

comparable results between reactions
performed in individual tubes or
in 4-plex reactions for a gene
quantification experiment.
Figure 2. Comparison of singleplex and multiplex gene expression assays. (A) EGFR assay,
FAM dye; (B) BRCA1 assay, VIC dye; (C) ESR1 assay, JUN dye; (D) RNase P assay, ABY dye.
Amplification was performed on the QuantStudio 7 Real-Time PCR System using TaqMan Multiplex
Master Mix. The figure shows amplification plots (left) and linear curves (right) for 4 assays amplified
in singleplex (blue) and 4-plex reactions (red) in a dilution series from 20,000 pg to 2 pg of reference
colon cDNA per 10 μL reaction. Average Ct value (lines) and average standard deviation (bars) for
the dilution series are represented in their respective graphs and show the concordance between
singleplex and 4-plex reactions. PCR efficiencies are: 96.09% for EGFR singleplex and 96.39%
for EGFR 4-plex; 93.56% for BRCA1 singleplex and 94.93% for BRCA1 4-plex; 97.13% for ESR1
singleplex and 95.81% for ESR1 4-plex; 96.91% for RNase P singleplex and 98.1% for RNase P
4-plex.
Improved probe performance A 0.5
0.5
0.5
0.5
B2M assay
B2M
B2M assay
B2M assay assay
30 30
30
B0.5
0.5
0.5
0.5
HPRT HPRT
assay assay
HPRT HPRT
assay assay 30 30
30
30 30
Introduction of ABY and JUN reporter 0.45
0.45
0.45
0.45 0.45
0.45
0.45
0.45
25 25
25 25 25
25
dyes and Mustang Purple passive 0.4
0.4
0.4
0.4 25 0.4
0.4
0.4
0.4 25

0.35 0.35
0.35 0.35 0.35
0.35
0.35 0.35
reference dye allows for optimal 20
20
20
20 20
20
20
20

dev.)

dev.)

dev.)

dev.)
(average)

(average)

(average)

(average)
0.3 0.3 0.3 0.3

(std.dev.)

(std.dev.)
CCt t(average)

CCt t(average)
(std.dev.)

(std.dev.)
0.3 0.3

(average)

(average)
0.3 0.3
4-color multiplex assays when used 0.25 0.25 15 15 0.25 0.25 15 15

CCt t(std.

CCt t(std.
0.25 0.25

(std.

(std.
0.25 15 15 0.25 15 15

with our FAM and VIC reporter dyes.

t t

t t
t t

t t
0.2 0.2 0.2 0.2

CC

CC
CC

CC
0.2 0.2 0.2 0.2
10 10
10 10 10
10
0.15 0.15
0.15 10 0.15 0.15
0.15 10
Please note that ABY and JUN 0.15
0.1 0.1
0.1
0.15
0.1 0.1
0.1
0.1 5 5 0.1 5 5
reporter dyes are available only with 5 5 5 5
0.05 0.05
0.05 0.05 0.05
0.05
0.05 0.05

QSY quencher, while FAM and VIC 0

0
0
0
FAM FAM
FAM VIC VIC
VIC ABY ABY
ABY JUN JUN
JUN
0
0
0
0
0
0
0
0
FAM FAM
FAM VIC VIC
VIC ABY ABY
ABY JUN JUN
JUN
0
0
0
0
FAM VIC ABY JUN FAM VIC ABY JUN
dyes are available with either MGB
GAPDH
GAPDH
assay assay
GAPDH RNaseP
RNaseP
assay assay
assay assay RNaseP
assay assay
or QSY quencher. A comparison with C 0.5
0.5
0.5
0.5
GAPDH 30
30
30
30 D0.5
0.5
0.5
0.5
RNaseP 30
30
30
30
0.45 0.45 0.45 0.45
a set of dyes from another supplier 0.45
0.4
0.45
0.4
25
25
25
25
0.45
0.4
0.45
0.4
25
25
25
25
0.4 0.4 0.4 0.4
shows that our combination of dyes 0.35
0.35
0.35
0.35 0.35
0.35
0.35
0.35
20 20
20 20 20
20
20 20
provides an earlier Ct for the majority dev.)

dev.)

dev.)

dev.)
(average)

(average)

(average)

(average)
0.3 0.3 0.3 0.3

(std.dev.)

(std.dev.)
CCt t(average)

CCt t(average)
(std.dev.)

(std.dev.)
(average)

(average)
0.3 0.3 0.3 0.3
0.25 0.25 0.25 0.25
of assays (Figure 3).
15 15 15 15
CCt t(std.

CCt t(std.
0.25 0.25
(std.

(std.
0.25 15 15 0.25 15 15

t t

t t
t t

t t
CC

CC
0.2 0.2 0.2 0.2
CC

CC
0.2 0.2 0.2 0.2
10 10
10 10 10
10
0.15 0.15
0.15 10 0.15 0.15
0.15 10
0.15 0.15

Optimized multiplex master mix 0.1

0.1
0.1
0.1
5 5
5
0.1
0.1
0.1
0.1
5 5
5
5 5
0.05 0.05 0.05 0.05
In multiplex PCR, it’s important to 0.05
0
0.05
0
0.05
0
0.05
0
0 0 0 0
0 0 0 0 0
0
0 0
have a robust master mix that allows FAM
FAM
FAM
FAM VIC
VIC
VIC
VIC ABY
ABY
ABY
ABY JUN
JUN
JUN
JUN FAM
FAM
FAM
FAM VIC
VIC
VIC
VIC ABY
ABY
ABY
ABY JUN
JUN
JUN
JUN

for amplification of each target in a
highly competitive environment. Our
new master mix composition was
developed to provide optimal multiplex
performance for each target in the
reaction. A comparison of our master
mix and a master mix from another
supplier in a 4-plex reaction shows an
Figure 3. Comparison of our new dye combination with a dye combination from another
supplier. Probes for (A) B2M, (B) HPRT, (C) GAPDH, and (D) RNase P gene expression assays were
synthesized with FAM, VIC, ABY, and JUN dyes with QSY quencher (green bars and diamonds) and
with another commercially available dye combination (blue bars and triangles). All possible gene-dye
combinations were tested. Reactions were prepared with TaqMan Multiplex Master Mix using 900 nM
of primer, 250 nM of probe, and 10 ng of cDNA. Amplification was performed on the QuantStudio
7 Real-Time PCR System using TaqMan Multiplex Master Mix. Bars represent average standard
deviation. Triangles and diamonds represent average Ct values.
A 0.50
0.50 35.00
35.00
B 0.60
0.60 35.00
35.00

earlier Ct for 3 of the targets amplified 0.50

0.50
0.45
0.45
35.00
35.00 0.60
0.60 35.00
35.00
33.00
33.00
0.45
0.45 0.50
0.50 33.00
33.00
with our new master mix and a lower 0.40
0.40
0.40
0.40
30.00
30.00
30.00
30.00
0.50
0.50
31.00
31.00
31.00
31.00
0.35
0.35 29.00
29.00
standard deviation for most of the 0.40
0.40
dev.)
dev.)
dev)

(average)

C (average)
dev)dev)

0.35
Ct(average)

Ctt(average)
0.35 25.00 29.00
29.00
0.30
0.30 25.00 0.40
0.40 27.00
27.00
dev.)
dev.)
(average)

Ct (average)
dev)

CCt (average)

Ct (average)
(std.

dilution points, demonstrating the

(std.

Ct (std.
Ct (std.

0.30
0.30 25.00
25.00 27.00
27.00
0.25
0.25 0.30
0.30 25.00
25.00
(std.
(std.

CCt (std.
CCt (std.

t
t
t

0.25
C

0.30
C

25.00
C

0.25 20.00 0.30 25.00

excellent performance of our 0.20
0.20 20.00 23.00
23.00
t
t
t

0.20
0.20 20.00
20.00 0.20
0.20 23.00
0.15
0.15 23.00
21.00
21.00
solution (Figure 4). 0.15
0.15
0.10
0.10 15.00
15.00
0.20
0.20
21.00
21.00
19.00
19.00
0.10
0.10
0.10
0.10 15.00
15.00 19.00
0.05 0.10 19.00
17.00
0.05 0.10 17.00
0.05
0.05 10.00
10.00 17.00
17.00
00 0.00
0.00 15.00
15.00
100,000
100,000 10,000
10,000 1,000
1,000 100
100 10
10 10.00
10.00 100,000
100,000 10,000
10,000 1,000
1,000 100
100 1010
00 0.00
0.00 15.00
15.00
100,000
100,000 Template
Templateper
10,000
10,000 perreaction
1,000reaction(pg)
1,000 100
(pg)
100 10
10 100,000
100,000 Template
10,000
Template
10,000 per
perreaction
1,000
1,000 reaction(pg)
100
(pg)
100 1010
Templateper
Template perreaction
reaction(pg)
(pg) Templateper
Template perreaction
reaction(pg)
(pg)
0.50 0.50
C 0.50
0.50
0.50
35.00
35.00
35.00
35.00
D 0.50
0.50
0.50
35.00
35.00
35.00
35.00
0.45
0.45 0.45
0.45
0.45
0.45
0.40 0.45
0.45
0.40
0.40 30.00
30.00 0.40 30.00
30.00
0.40
0.40
0.35 30.00
30.00 0.40
0.40
0.35 30.00
30.00
0.35 0.35
dev.)
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dev)

(average)

C (average)
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CCt t(average)

Ct t(average)
0.35
0.35
0.30 25.00 0.35
0.35
0.30 25.00
dev.)

25.00 25.00
dev.)

0.30 0.30
(average)

Ct (average)
dev)

(std.
Ctt (average)

Ct (average)
(std.

CCt t(std.
CCt t(std.

0.30
0.30
0.25 25.00
25.00 0.30
0.30
0.25 25.00
25.00
0.25 0.25
(std.
Ctt (std.
(std.
Ctt (std.

0.25
0.25
0.20 20.00 0.25
0.25
0.20 20.00
0.20 20.00 0.20 20.00
C
C
C

0.20
0.20 20.00
20.00 0.20
0.20 20.00
20.00
0.15
0.15 0.15
0.15
0.15
0.15
0.10 15.00 0.15
0.15
0.10 15.00
0.10 15.00 0.10 15.00
0.10
0.10 15.00
15.00 0.10
0.10 15.00
15.00
0.05
0.05 0.05
0.05
0.05
0.05
00 10.00
10.00 0.05
0.05
00 10.00
10.00
00 100,000
100,000 10,000
10,000 1,000
1,000 100
100 10
10 10.00
10.00 00 100,000
100,000 10,000
10,000 1,000
1,000 100
100 1010 10.00
10.00
100,000
100,000 Template
Templateper
10,000
10,000 reaction
1,000
per reaction(pg)
1,000 100
100
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10 100,000
100,000 Template
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1,000
1,000
Template reaction(pg)
100
100
(pg) 1010
Templateper
Template perreaction
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Template perreaction
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(pg)

Figure 4. Comparison of TaqMan Multiplex Master Mix with another commercially available
master mix. (A) B2M assay, FAM dye; (B) RNase P assay, VIC dye; (C) GAPDH assay, ABY dye; (D)
HPRT assay, JUN dye. All assays used QSY quencher. The graph shows average standard deviation
(bars) and average Ct values (cross and triangle) for 4-plex reactions using a dilution series from
100 ng to 10 pg of cDNA per 10 μL reaction. All amplifications were performed on the ViiA 7 Real-
Time PCR System using the cycling conditions recommended for each master mix. Green represents
TaqMan Multiplex Master Mix, and blue represents 4-plex reactions with another commercially
available master mix.
Optimized to minimize development time. A new multiplex References
time-to-results PCR user guide was developed to 1. Multiplex PCR User Guide. Available at thermofisher.
com/multiplexqpcr
Developing a multiplex PCR assay guide you through the development 2. TaqMan multiplex qPCR: Accurate, sensitive, and as
requires time to correctly design the and optimization process [1], and efficient as traditional singleplex qPCR. Application note
assay and optimize the reaction. our custom services will allow you available at lifetechnologies.com/multiplexqpcr

Using our complete solution, for which to delegate assay design to our
all components were developed to experienced team to minimize your
work together, helps increase your efforts.
chances of success and limits your

Ordering information

Product Cat. No.

TaqMan QSY probes

TaqMan QSY Probe, 6,000 pmol 4482777

TaqMan QSY Probe, 20,000 pmol 4482778

TaqMan QSY Probe, 50,000 pmol 4482779

Control kits
TaqMan GAPDH Assay, JUN-QSY 20X 4485712

TaqMan GAPDH Assay, JUN-QSY PL 20X 4485713

TaqMan RNaseP Assay, ABY-QSY 20X 4485714

TaqMan RNaseP Assay, ABY-QSY PL 20X 4485715

Multiplex master mixes

TaqMan Multiplex Master Mix, 1 mL 4461881

TaqMan Multiplex Master Mix, 5 mL 4461882

TaqMan Multiplex Master Mix, 50 mL 4486295

Other formats are available at lifetechnologies.com/multiplexqpcr

Calibration plates
96-Well Calibration Plate, Mustang Purple dye 4461599

96-Well Calibration Plate, JUN dye A24737

96-Well Calibration Plate, ABY dye A24738

Calibration plates are also available for 96-well Fast and 384-well plate formats.
Visit thermofisher.com/multiplexqpcr for more information.

Find out more at thermofisher.com/multiplexqpcr

For Research Use Only. Not for use in diagnostic procedures. © 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks
are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a registered trademark of Roche
Molecular Systems, Inc., used under permission and license. COL12035 0616