F2143K

Instruction manual for THUNDERBIRD Next Probe qPCR Mix

THUNDERBIRDTM Next Probe qPCR Mix
QPX-101T
QPX-101

1 mL x 1
1.67 mL x 3

Store at -20°C, protected from lightContents[1]

Introduction[2]

Components[3]

Primer/probe design[4]Template DNA[5]

Protocol
1. Standard reaction set up
2. Cycling conditions
2-1. Real-time PCR conditions using Applied Biosystems StepOnePlus™
2-2. LightCycler® 96
2-3. CFX96 Touch™ Deep Well

[6]

Optimization of conditions[7]

Related Protocol
cDNA synthesis[8]

Troubleshooting[9]

Related products

CAUTIONAll reagents in this kit are intended for research purposes only. Do not use for diagnostic or clinical purposes. Please observe
general laboratory precautions and observe safety procedures while using this kit.
-TaqMan® and LightCycler® are registered trademarks of Roche Diagnostic Systems, Inc.
-SYBR® is a registered trademark of Thermo Fisher Scientific, Inc.

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[1] Introduction

Description
THUNDERBIRDTM Next Probe qPCR Mix is a highly efficient 2× Master Mix forreal-time PCR using TaqMan® probes. The master mix contains all required components,
except for ROX reference dye, probe and primers (50x ROX reference dye is individually
supplied with this kit). The master mix facilitates reaction setup, and improves the
reproducibility of experiments. This product is an improved version of
THUNDERBIRDTM Probe qPCR Mix (Code No. QPS-101). This kit is optimized for
multiplexing and crude samples containing PCR inhibitors.

Features-High specificity
The specificity for the detection of low-copy targets is improved.
-Homogeneous amplification
The dispersion of PCR efficiency between targets is reduced by a new PCR enhancer.
-Broad dynamic range
High specificity and effective amplification enable the detection of a broad dynamic
range.
-Multiplex detection
Multiple targets can be detected using TaqMan® probes labeled with different
fluorescent dyes in one reaction.
-Tolerant of PCR inhibitors
This kit can reduce the inhibition by PCR inhibitors such as hematin.
-Fast PCR
This reagent enables amplification using fast cycle condition.
-Hot start PCR
The master mix contains anti- DNA polymerase antibodies for hot start technology.
The antibodies are easily inactivated in the first denaturation step, thereby activating
the DNA polymerase.
-Utilization of dUTP
This master mix contains dUTP instead of dTTP. Therefore, the rate of false-positive
detection can be reduced by adding uracil-N-glycosylase (UNG). *UNG is not
supplied with this kit. Uracil-DNA Glycosylase (UNG), Heat-labile (Code No.
UNG-101) can be used.
-Compatibility for various real-time cyclers.
The reagent is applicable to most real-time cyclers (i.e. Block type and glass capillarytype). Because the 50x ROX reference dye is individually supplied with this kit, the kit
can be applied to real-time cyclers that require a passive reference dye.

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[2] Components

This kit includes the following components for 100 reactions (QPX-101T) and 500
reactions (QPX-101), with 20 l per reaction. All reagents should be stored at -20°C.
<QPX-101T>
THUNDERBIRDTM Next Probe qPCR Mix
50x ROX reference dye

1 ml x 1
50 l x 1

<QPX-101>
THUNDERBIRDTM Next Probe qPCR Mix
50x ROX reference dye

1.67 ml x 3
250 l x 1

Notes:-THUNDERBIRDTM Next Probe qPCR Mix can be stored, protected from light, at 2-8°C
for up to 3 months. For longer storage, this reagent should be kept at -20°C andprotected from light. No negative effect was detected by 10 freeze-thaw cycles of
THUNDERBRIDTM Next Probe qPCR Mix. This reagent does not contain the ROX
reference dye.
-50x ROX reference dye can be stored, protected from light, at 2-8°C or -20°C. For
real-time cyclers that require a passive reference dye, this reagent must be added to thereaction mixture at a concentration of 1x or 0.1x. The master mix solution with the ROX
reference dye can be stored, protected from light, at 2-8°C for up to 3 months. For longer
storage, this reagent should be kept at -20°C and protected from light. The pre-mixed
reagents can be prepared according to the following ratios. [5] Table 1 shows the optimal
concentration of the ROX dye.
1x solution
THUNDERBIRDTM Next Probe qPCR Mix : 50x ROX reference dye= 1.67 ml : 66.8 l
THUNDERBIRDTM Next Probe qPCR Mix : 50x ROX reference dye= 1 ml : 40 l0.1x solution
THUNDERBIRDTM Next Probe qPCR Mix : 50x ROX reference dye = 1.67 ml : 6.7 l
THUNDERBIRDTM Next Probe qPCR Mix : 50x ROX reference dye = 1 ml : 4 l
For real-time cyclers that do not require a passive reference dye, THUNDERBIRDTM
Next Probe qPCR Mix without the ROX reference dye can be used.

[3]

Primer/probe
design1. Primer conditions
Highly sensitive and quantitative data depend on primer design. The primer should bedesigned according to the following suggestions;-Primer length: 20-30 mer-GC content of primer: 40-60%-Target length: ≤ 200 bp (optimally, 80-150 bp)-Melting temperature (Tm) of primers: 60-65°C

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-Purification grade of primers: Cartridge (OPC) grade or HPLC grade
Notes:-Longer targets (>200 bp) reduce efficiency and specificity of amplification.
-Tm of the primers can be flexible, because the Tm value depends on the calculation
formula.

2. Fluorescent probe
The probes should be designed according to the guidelines of each probe system.
Because insufficiently purified probes may inhibit the reaction, HPLC-grade probes
should be used.

[4]

Template DNA

The following DNA samples can be used as templates.

1. cDNA
Non-purified cDNA, generated by reverse transcription reactions, can be used directly forreal-time PCR using THUNDERBIRDTM Next Probe qPCR Mix. Up to 10% of thevolume of a cDNA solution can be used for a real-time PCR reaction. However, excess
volume of the cDNA may inhibit the PCR. Up to 20% (v/v) of the cDNA solution fromthe ReverTra AceTM qPCR RT Kit (Code No. FSQ-101), the ReverTra AceTM qPCR RT
Master Mix (Code No. FSQ-201) and the ReverTra AceTM qPCR RT Master Mix with
gDNA remover (Code No. FSQ-301) can be used for real-time PCR.

2. Genomic DNA, Viral DNA
Genomic DNA and viral DNA can be used at up to 200 ng in 50 L reactions.

3. Plasmid DNA
Although super-coiled plasmids can be used, linearized plasmid DNA produces moreaccurate assays. The copy number of the plasmid DNA can be calculated by thefollowing formula.
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[5]Protocol1.

Standard reaction set up

Reagent
Sterilized water

Reaction volume
50 µl
20 µl
X µl
X µl

Final
Concentration

THUNDERBIRDTM Next Probe qPCR Mix

25 µl

10 μl

1x

Forward Primer
Reverse Primer

15 pmol
15 pmol

6 pmol6 pmol

0.3 μM*1
0.3 μM*1

TaqMan® Probe

10 pmol

4 pmol

0.2 μM*2

50x ROX reference dye

1μl / 0.1 μl 0.4μl / 0.04μl 1x / 0.1x*3(Uracil-N-glycosylase)[optional]

1 unit*4

0.4 units*4

DNA solution
Total

Y µl
50 µl

Y µl
20 µl

Notes:*1 If the expected results are not obtained at 0.3 µM, [6] Optimization of conditions canbe consulted. The same conditions can be applied in the case of multiplex detection.
*2 If the expected results are not obtained at 0.2 µM, [6] Optimization of conditions canbe consulted. The same conditions can be applied in the case of multiplex detection.
*3 50× ROX Reference dye must be added when using real-time cyclers that require a
passive reference dye, according to Table 1. Table 1 shows the optimum concentrationof the ROX Reference dye. This dye is not necessary for real-time cyclers that do not
require a passive reference dye.
Table 1 Recommended ROX dye concentration
Real-time cycler
Applied Biosystems 7000, 7300, 7700, 7900HT
StepOne™, StepOnePlus™, etc.
Applied Biosystems 7500, 7500Fast, QuantStudio™
Agilent cyclers (Optional), etc.
Roche cyclers, Bio-Rad cyclers, Qiagen cyclers, etc.

Optimal dye concentration(dilution ratio)
1× (50:1)
0.1× (500:1)
Not required*4 Heat-labile uracil-N-glycosylase (UNG) should be used. UNG is not supplied withthis kit. It is recommended that you use Uracil-DNA Glycosylase (UNG), Heat-labile(UNG-101) optionally.

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2. Cycling conditions
The following table shows the recommended thermal conditions using primers designed
according to the recommended primer and probe conditions described in [3] Primer/probe
design. These conditions can be applied to multiplex assays.
Fast cycling mode
<2-step cycle>

Temperature

Time

Ramp

(UNG treatment)(20–25°C*1)(10 min*1)(Maximum)Pre-denaturation

95°C

20 secMaximum

Denaturation:

95°C

5 sec

Maximum

Extension:

60°C
10 secMaximum(data collection should be set at the extension step)

40 cycles*2Standard cycling mode
<2-step cycle>

Temperature

Time

Ramp

(UNG treatment)(20–25°C*1)(10 min*1)(Maximum)Pre-denaturation

95°C

20 secMaximum

Denaturation:

95°C

5 sec

Maximum

Extension:

60°C
30 secMaximum(data collection should be set at the extension step)

40 cycles*2*1 [Optional] The indicated temperature and time are typical conditions for UNG. The
conditions can be optimized according to the particular instruction manual from the
supplier of UNG. Uracil-DNA Glycosylase (UNG), Heat-labile (Code No. UNG-101)
can be used.
*2 The number of cycles can be increased up to 45.

2-1. Real-time PCR conditions using Applied Biosystems StepOnePlus™
(normal block type, software version 2.3)
The following is an example of a TaqMan® assay using Applied Biosystems
StepOnePlus™. These conditions can also be useful for cyclers such as the Applied
Biosystems 7500 Fast Real-Time PCR System and QuantStudio™ Real-Time PCR
System.

(1) Select “Design Wizard”, “Advanced Setup” or “QuickStart” after starting thesoftware.

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(2) ” Select “TaqMan® Reagents” as reagents in the following tabs.
Design Wizard
Advance Setup
QuickStart

Methods & Materials
Setup  Experiment Properties
Experiment Properties(3) Select “Run Methods” and input the reaction volume at “Reaction Volume Per
Well”.

(4) Select “Holding Step” and input 95°C, 20 s.
(5) Select “Cycling Stage” and input 95°C, 5 s, 60°C, 10 s, 40 cycles.

*1*2*1 Input of actual reaction volume is important to achieve a successful analysis.
*2 Set the data collection at the extension step. Some real-time cyclers or software needover 25 sec for the extension step. In these cases, the time should be set according toeach instruction manual (e.g. Applied Biosystems QuantStudio™ 5, 96-well, 0.2mL:≥ 25 sec; Applied Biosystems 7000/73000: ≥ 31 sec; Applied Biosystems 7500:≥ 35 sec.).
(6) Start the program after setting the plate or tubes.

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2-2. LightCycler® 96(software version 1.1)
The following is an example of a TaqMan® assay using the Roche LightCycler® 96.

(1) Select “Create New Experiment”(2) Input reaction volume.
(3) Select Preincubation Two-Step Amplification at “Predefined Program” in the“Run Editor” tab.
(4) Select the first “Preincubation” and input 95°C as “Target” and 60 s as “Duration”.
(5) Select the second “2 Step Amplification” and input 95°C as “Target” and 5 s as
“Duration” followed by 60°C as “Target” and 10 s as “Duration”.

*1*2*1 Input of the actual reaction volume is important for a successful analysis.
*2 Set the data collection at the extension step.
(6) Insert the PCR tubes or plate.
(7) Start the program.

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2-3. CFX96 Touch™ Deep Well
(software version 3.1)
The following is an example of a TaqMan® assay using the Bio-Rad
CFX96 Touch™ Deep Well.

(1) Select “User-defined”.
(2) Select “Create New …” and input reaction volume.
(3) Input the following conditions: 95°C, 20 s, 95°C, 5 s, 60°C, 10 s, 40 cycles.

*1(4) Start the program after setting the plate or tubes.

*2*1 Input of the actual reaction volume is important for a successful analysis.
*2 Select “Add Plate Read to Step” and set the data collection point at the extension step.
(4) Insert the PCR tubes or plate.
(5) Start the program.

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[6] Optimization of conditions
1. Optimization procedure
If the expected performance is not obtained using the standard conditions ([5] Protocol),the reaction conditions should be optimized. The low sensitivity is caused by poor PCR
efficiency or failure of detection with TaqMan® probe. After confirming that the primers
and TaqMan® probes have been designed appropriately, the extension (annealing)
temperature and time should be optimized according to the following flow chart. If thedesigns of primers and TaqMan® probes seem to be inappropriate, they should beredesigned based on [3] Primer/probe design.
Low sensitivity↓

Optimization of extension (annealing) temperature and time (see [6] 2)↓

Optimization of concentrations of primers and TaqMan® probes (see [6] 3)

2. Optimization of extension (annealing) temperature and time
Decrease or prolonging of extension (annealing) temperature or time can improve the
PCR efficiency in cases in which the annealing of the primer or TaqMan® probe isinsufficient. In cases in which a decrease of extension (annealing) temperature does not
improve the sensitivity, nonspecific amplification such as primer–dimer formation may
reduce the PCR efficiency. In such cases, an increase of the extension (annealing)
temperature may increase the efficiency.
In cases in which changes of the extension (annealing) temperature and time do not
improve the results, the concentration of primers or TaqMan® probes should be optimized
according to [6] 3.
<Flow of optimization of extension (annealing) step>60°C, 10-30 s (standard conditions)↓~55°C, 10-30 s↓Tendency for improvement~55°C, 60 s

↓No improvement~65°C, 10-30 s

Notes
The number of cycles can be increased up to 45.
VeriFlex™ of Applied Biosystems StepOnePlus™ or Temperature gradient of Bio-Rad cyclers facilitates
optimization of the extension (annealing) temperature.

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3. Optimization of concentrations of primers and TaqMan® probes(1) Increase primer and/or TaqMan® probe concentration
Increasing the primer concentration can improve the efficiency in cases in which theannealing of primers is insufficient. Similarly, increasing the TaqMan® probe
concentration can increase the sensitivity in cases in which the annealing of TaqMan®
probes is insufficient.
First, the primer concentration should be optimized between 0.3 and 0.5 µM with the
TaqMan® probe concentration at 0.2 µM. Then, the concentration of TaqMan® probeshould be increased up to 0.4 µM in cases in which the increase of primers has not
improved the results.
(2) Decrease primer concentration
Decreasing the primer concentration may improve the PCR efficiency in cases withprimer–dimer formation. In such cases, the primer concentration should be decreased
between 0.2 and 0.3 µM with the TaqMan® probe concentration at 0.2 µM.
Please note that a low concentration of TaqMan® probe of less than 0.2 µM decreases thesensitivity because of low fluorescence intensity.

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[7]Related Protocol

cDNA synthesis
cDNA synthesized by various cDNA synthesis reagents can be used with
THUNDERBIRDTM Next Probe qPCR Mix. However, cDNA synthesized by a reagent
specialized for real-time PCR can increase sensitivity.
ReverTra Ace™ qPCR RT Kit (Code No. FSQ-101), ReverTra Ace™ qPCR RT MasterMix (Code No. FSQ-201) and ReverTra Ace™ qPCR RT Master Mix with gDNA
Remover (Code No. FSQ-301) are cDNA synthesis kits suitable for real-time PCR.
Here, the protocol with ReverTra Ace™ qPCR RT Master Mix and ReverTra Ace™
qPCR RT Master Mix with gDNA Remover is described. However, for the detailed
protocol, please refer to the instruction manual of the kit.
ReverTra Ace™ qPCR RT Master Mix (Code No. FSQ-201)(1) Denaturation of RNA [optional]
Incubate the RNA solution at 65°C for 5 min, and then keep on ice.
Notes:-This step increases the efficiency of reverse transcription of RNA templates that form
secondary structures.
-This step should be performed before adding 5x RT Master Mix.
(2) Preparation of the reaction solution
Prepare the following reagents on ice.
5x RT Master Mix
RNA template

2 L
1 pg – 1 g

Nuclease-free Water

X LTotal Volume

10 L(3) Reverse transcription reaction-Incubate at 37°C for 15 min.
-Incubate at 50°C for 5 min. [optional]-Heat to 98°C for 5 min.
-Store the reacted solution* at 4°C or – 20°C
*This solution can be used in the real-time PCR reaction directly or after dilution.
Notes:-ReverTra Ace™ excels at high reaction temperatures (up to 50°C). This step may
increase the efficiency of the reverse transcription.

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ReverTra Ace™ qPCR RT Master Mix with gDNA Remover (Code No. FSQ-301)(1) Preparation of the “4x DN Master Mix” and “gDNA Remover” mixture.
Prior to use, a 1 in 50 volume of gDNA remover should be added to 4x DN
Master Mix (e.g. 4x DN Master Mix : gDNA remover = 440 L : 8.8 L).
Notes
4x DN Master Mix with gDNA remover can be stored at -20°C for at least for 3 months. The mixture canbe prepared in a smaller volume [e.g. 4x DN Master Mix : gDNA remover = 220 L : 4.4 L].

(2) Denaturation of RNA [optional]
Incubate the RNA solution at 65°C for 5 min, and then keep on ice.
Notes
-This step increases the efficiency of reverse transcription of RNA templates that form
secondary structures.
-This step should be performed before adding 4x DN Master Mix.

(3) Preparation of the DNase I reaction solution:
Prepare the following reagents on ice.
2 L

4x DN Master Mix
RNA template

0.5 pg – 0.5 gNuclease-free Water

X LTotal Volume

8 L(4) Incubate at 37°C for 5 min.
(5) Preparation of the for reverse transcription solution;
Prepare the following reagents on ice.
Reacted solution from (4)8 L5x RT Master Mix II

2 LTotal Volume

10 L(6) Reverse transcription reaction-Incubate at 37°C for 15 min.
-Incubate at 50°C for 5 min. [optional]-Heat to 98°C for 5 min.
-Store the reacted solution* at 4°C or – 20°C
*This solution can be used in the real-time PCR reaction directly or after dilution.

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Notes:-ReverTra Ace™ excels at high reaction temperatures (up to 50°C). This step may increase
the efficiency of the reverse transcription.

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[8]Troubleshooting
Symptom
Loss of linearity in thehigh cDNA/DNA
concentration region.

Cause

Solution

Inhibition by the components in the
cDNA/DNA solution.

-DNA: The DNA sample may contain PCR
inhibitors. The DNA samples should be
repurified.
-cDNA: The components in the cDNA synthesis
reagent may inhibit the PCR reaction. The cDNA
sample should be used after dilution.
When the DNA/cDNA copy number is lower than
10 copies per reaction, the linearity of the reaction
tends to be lost. The template concentration
should be increased.
The diluted DNA templates tend to be absorbed
onto the tube wall. Dilution should be performed
just prior to experiments.
In the probe assay, primer dimers are not detected.
However, dimer formation may reduce theamplification efficiency of the target, especiallyfor reactions at low template concentration. The
reaction conditions should be optimized or theprimer sequences should be changed.
In the probe assay, non-specific amplification is
not detected. However, non-specific amplification
may reduce the amplification efficiency of thetarget. The reaction conditions should be
optimized or the primer sequences should bechanged.
Optimize the cycling conditions according to [5].
Fresh primer solution should be prepared.
The Ct value on the linear region should be used
to calculate PCR efficiency.
The Ct value on the linear region should be used
to calculate PCR efficiency.

The template DNA is insufficient.

Lost of linearity or
lower signal in the low
DNA/cDNA
concentration region.

Adsorption of the DNA to the tube
wall.
Competition with primer dimer
formation.

Loss of linearity of theamplification carves.

Competition with non-specificamplification.

The PCR efficiency islower than 90%(slope: <-3.6)

Inappropriate cycling conditions.
Degradation of the primers.
The calculation of the PCR
efficiency is inappropriate.
The calculation of the PCR
efficiency is inappropriate.

The PCR efficiency ishigher than 110%(slope: >-3.1)
Reproducibility is not
good.

Poor purification of the template
DNA.
Absorption of the template DNA to
the tube wall.
Plasmid DNA or PCR product isused as a template.

Inappropriate thermal conditions.
Low purity of the primers or probes.

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Low-purity DNA may contain PCR inhibitors.
Re-purify the DNA samples.
Diluted DNA templates tend to be absorbed onto
the tube wall. Dilution of the template
DNA/cDNA should be performed just prior toexperiments.
In general, plasmid DNA or PCR product is used
at low concentration. Diluted DNA templates tend
to be absorbed onto the tube wall. Dilution of thetemplate DNA/cDNA should be performed justprior to experiments. Dilution with a carrier
nucleic acid solution (Yeast RNA) is also
effective in improving linearity.
Optimize the thermal conditions according to [5].
Different lots of primers or probes may show
different results. When the lot is changed, priortesting of the primer or probe should be
performed.

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SymptomCause

Amplification from thenon-template control(NTC).

Contamination or carry over of the
PCR products.
Inappropriate settings offluorescence measurement, such as
in the case of multiplex PCR.

Low amplification
curve signal /Unstable amplification
curve signal.

Excessive amount of ROX referencedye.
Inappropriate settings offluorescence measurement.
Low purity of fluorescent probes.
Excessive intensity of the quencher
Dye.
Degradation of the probe.
Insufficient fluorescencemeasurement time.
Insufficient reaction volume.

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Solution
Change the contaminated reagent.
In multiplex experiments, inappropriate setting offluorescence measurement may cause thedetection of noise by the cross talk of fluorescentdyes. Settings should be reconfirmed.
Excessive amount of ROX reference dye may
cause low signal. 50x ROX reference dye should
be used according to [5] Table 1.
Settings should be confirmed according to the
instruction manual of each detector.
Low purity of the probe may increase the baseline. HPLC grade probes should be used.
Certain quenchers (e.g. TAMRA) may cause ahigher baseline because of its fluorescence. Useof a non-fluorescent quencher may improve thehigh baseline.
Store the probes according to the manufacture’s
recommendations.
Certain detection systems require a longer time todetect the fluorescent signal. Longer extension(measurement) time (45-60 sec) may improve theunstable signal.
Low reaction volume may cause an unstablesignal. Increase the reaction volume.

CHINA
TOYOBO Bio-Technology, CO., LTD.
Tel.: (86)-21-58794900.4140

FOR RESEARCH USE ONLY.

15
NOT FOR HUMAN OR DIAGNOSTIC USE.

[9]Related products
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Heat-labile Uracil-DNA Glycosylase

Uracil-DNA Glycosylase (UNG), Heat-labile

JAPAN
TOYOBO CO., LTD.
Tel(81)-6-6348-3846
www.toyobo.co.jp/e/bio
bio_overseas@toyobo.jp

CHINA
TOYOBO Bio-Technology, CO., LTD.
Tel.: (86)-21-58794900.4140

FOR RESEARCH USE ONLY.

16
NOT FOR HUMAN OR DIAGNOSTIC USE.