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Ｆ1469Ｋ

Instruction manual for THUNDERBIRD Probe One-step qRT-PCR Kit 1602

THUNDERBIRD® Probe One-step qRT-PCR Kit
QRZ-101

100 reactions

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

Introduction[2]

Components[3]

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

Protocol
1. Reaction mixture setup
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]

Application data

[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 Molecular Systems, Inc.

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www.toyobo.co.jp/e/bio[1] Introduction

Description
THUNDERBIRD® Probe One-step qRT-PCR Kit is a one-step real-timereverse-transcription polymerase chain reaction (RT-PCR) kit using the highly efficient
reverse transcriptase “ReverTra Ace®” and Tth DNA polymerase as a PCR enzyme. This
product can be used mainly in TaqMan® probe assays. The one-step system is suitable forhigh-throughput analysis because of its simple reaction setup. In addition, this system canreduce the risk of cross-contamination.
The combination of the two enzymes and optimized buffer system enable the effective
detection and quantification of a small amount of RNA. This kit can also detect various
kinds of RNA with different sequences because it is tolerant of target sequence diversity.

Features-Rapid and highly sensitive
THUNDERBIRD® Probe One-step qRT-PCR Kit can achieve the rapid and highly
sensitive quantification of a small amount of RNA by a one-step qRT-PCR method withprobes. This kit is suitable for the quantification of RNA viruses or mRNA expressed ata low level.
-Tolerant of target sequence diversity
This kit can detect various types of RNA efficiently because of its tolerance of thediversity of target sequences.
-Tolerant of PCR inhibitors
This kit can reduce the inhibition by PCR inhibitors such as hematin.
-Utilization of dUTP
This kit contains dUTP instead of dTTP in 2× Reaction Buffer. Therefore, the rate offalse-positive detection can be reduced by adding uracil-N-glycosylase (UNG).
*UNG is not supplied with this kit.
-Multiplex detection
Multiple targets can be detected using TaqMan® probes labeled with different
fluorescent dyes in one reaction.
-Hot start system using antibodies
Hot start technology, using anti-DNA polymerase antibodies, allows highly specific andreproducible amplification. The antibodies are easily inactivated in the firstdenaturation step, minimizing the damage to DNA polymerase.
-Compatibility with various real-time cyclers
The reagent is applicable to most real-time cyclers (i.e., block type and glass capillarytype). Because the 50× ROX Reference dye is individually supplied with this kit, thekit can be applied to real-time cyclers that require a passive reference dye.

[2] Components

This kit includes the following components for 100 reactions, with 50 µl per reaction. All
reagents should be stored at −20°C.
2× Reaction Buffer
DNA Polymerase
RT Enzyme Mix
50× ROX Reference dye
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2 × 1.25 ml
125 µl125 µl100 µl2 × 1.25 ml

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Notes:-2× Reaction Buffer contains essential components for the reaction (buffer, salts, dATP,
dCTP, dGTP, and dUTP, etc.).
-DNA Polymerase is a mixture of Tth DNA polymerase and hot start antibodies.
-RT Enzyme Mix contains ReverTra Ace® as a highly efficient reverse transcriptase andRNase inhibitor.
-50× ROX Reference dye can be used as a passive reference in real-time cyclers from
Applied Biosystems® and Agilent Technologies to compensate for the variation ofreaction volumes between tubes. The final concentration of ROX dye should be changed
according to the real-time cycler. In the case of cyclers that do not require compensation,
ROX dye does not need to be added (see [5] Protocol).
-RNase free water should be used for preparation of the reaction mixture.

[3] Primer/probe
design1. Design of primers
Highly sensitive and quantitative data depend on the primer design. The primer should
be designed according to the following suggestions:-Primer length: 18–25-mer-GC content of primer: 40–60%-Melting temperature (Tm) of primer: 60–65°C-Purification grade of primer: Cartridge (OPC) grade or high-performance liquid
chromatography (HPLC) grade-Target length: 70–200 bp
Notes:-The reverse primer can also work as a primer for reverse transcriptase in this kit.
Random or oligo dT primer is not required.
-Larger targets (>200 bp) tend to reduce the efficiency and specificity of amplification.
-Amplification from genomic DNA can be avoided by designing the primers on
different exons or exon junctions.

2. Design of TaqMan® probes
Because insufficiently purified probes may inhibit the reaction, HPLC-grade probes
should be used. TaqMan® probe should be designed according to the following
suggestions:-Probe length: 20–30-mer-GC content of probe: 40–60%-Tm of probe: 65–70°C-Purification grade of primers: HPLC grade

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Notes:-Detection of an amplicon from genomic DNA can be avoided by designing the
TaqMan® probe on an exon junction.
The Tm of primers and probes should be calculated using the nearest neighbor method.
The Tm values in this manual were calculated using this method with the following
parameters: Na+ concentration, 50 mM; and oligonucleotide concentration, 0.5 μM.

3. Checking the performance of primers and TaqMan® probes
The performance of primers and TaqMan probes can be checked using the following
experiments.
(1) Prepare a dilution series with three or more dilutions of template RNA (e.g., 0.2
ng/µl, 2 ng/µl, 20 ng/µl).
(2) Perform qRT-PCR assay using the diluted RNA (5µl in 20-µl reaction) with the
newly designed primers and TaqMan® probe, and draw a standard curve according
to [5] Protocol.
(3) Confirm that the PCR efficiency is between 85% and 115% and R2 is equal to or
greater than 0.98.
If the PCR efficiency and/or R2 are outside of these ranges, the reaction conditions shouldbe optimized according to [6] Optimization of conditions. If this does not improve theresult, the primers and/or TaqMan® probe should be redesigned.

[4] Template RNA

The following RNAs are appropriate for highly efficient detection and quantification.
(1) Total RNA
Total RNA (e.g., the acid-guanidium-phenol-chloroform method or the spin column
method) can be used directly as a template with this kit. Total RNA usually contains
1–2% mRNA.
(2) Poly(A)+ RNA (mRNA)
Poly(A)+ RNA is useful to detect low-abundance mRNA. However, poly(A)+ RNA
should be treated carefully because it is more sensitive to RNase than total RNA.
(3) Viral RNA
Viral RNA purified by various methods (e.g., the acid-guanidium-phenol-chloroform
method or the spin column method) can be used.

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Protocol1.

Reaction mixture setup
Reaction volume

Final

50-µl reactio 20-µl reaction

Concentration

RNase free water

X µl

X µl

2× Reaction buffer

25 µl

10 μl

DNA Polymerase

1.25 µl

0.5 μl

RT Enzyme Mix

1.25 µl

0.5 μlForward primer

25 pmol

10 pmol

0.5 μM*1

Reverse primer

25 pmol

10 pmol

0.5 μM*1

10 pmol

4 pmol

0.2 μM*2

1μl / 0.1 μl

0.4 μl / 0.04 μl

1× / 0.1×*3(Uracil-N-glycosylase)*4 [optional]

1 unit

0.4 units

RNA solution

Y µl*5Y µl*5

50 µl

20 µl

®

TaqMan probe
50× ROX Reference dye

1×

1 unit / 50 µl*1 If the expected results are not obtained at 0.5 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,
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. The amount of UNG can bechanged according to the recommendations from each supplier. UNG is not suppliedwith this kit.
*5 The concentration of total RNA in a reaction can be increased up to 25 ng/µl. An
excessive amount of RNA inhibits the reaction.

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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.

(UNG treatment)
Reverse transcription
Pre-denaturation
Denaturation
Extension (annealing)

Temperature
Time
Ramp
(20–25°C*1)(10 min*1)(Maximum)
50°C
10 minMaximum
95°C
60 s
Maximum
95°C
15 s
Maximum
60°C
45 s
Maximum(Data collection should be performed at theextension step.)

40 cycles*2*1 [Optional] The uracil-N-glycosylase (UNG) treatment step should be added before thecDNA synthesis step. The indicated temperature and time are typical conditions for
UNG. The conditions can be optimized according to the particular instruction manualfrom the supplier of UNG.
*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.
(1) Select “Design Wizard”, “Advanced Setup” or “QuickStart” after starting thesoftware.
(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 “Add Step” and input 50°C, 10 min.
(5) Select “Holding Step” and input 95°C, 60 s.
(6) Select “Cycling Stage” and input 95°C, 15 s, 60°C, 45 s, 40 cycles.
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www.toyobo.co.jp/e/bio(7) Start the program after setting the plate or tubes.

*1*2*1 Input of the actual reaction volume is important for a successful analysis.
*2 Set the data collection at the extension step.

2-2. LightCycler® 96(software version 1.1)
(1) Select “Create New Experiment”(2) Input reaction volume.
(3) Select Preincubation Preincubation Two-Step Amplification at “Predefined
Program” in the “Run Editor” tab.
(4) Select the first “Preincubation” and input 50°C as “Target” and 600 s as “Duration”.
(5) Select the second “Preincubation” and input 95°C as “Target” and 60 s as
“Duration”.
(6) Select the second “2 Step Amplification” and input 95°C as “Target” and 15 s as
“Duration” followed by 60°C as “Target” and 45 s as “Duration”(7) Start the program after setting the plate or tubes.

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*2

*1

*3

*1 Input of the actual reaction volume is important for a successful analysis.
*2 The input screen consists of initial denaturation and two subsequent steps by default.
The cDNA synthesis step should be added by selecting “+”.
*3
Set the data collection at the extension (annealing) step.

2-3. CFX96 Touch™ Deep Well
(software version 3.1)
(1) Select “User-defined”.
(2) Select “Create New …” and input reaction volume.
(3) Select “Insert Step” and input 50°C and 10 min.
(4) Start the program after setting the plate or tubes.
(5) Input the following conditions: 95°C, 60 s, 95°C, 15 s, 60°C, 45 s, 40 cycles.
(6) Start the program after setting the plate or tubes.

*1

*2

*3

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www.toyobo.co.jp/e/bio*1 Input of the actual reaction volume is important for a successful analysis.
*2 The input screen consists of initial denaturation and two subsequent steps by default.
The cDNA synthesis step should be added by selecting “Insert Step”.
*3
Set the data collection at the extension (annealing) step.

[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, 45 s (standard conditions)↓~55°C, 45 s

↓Tendency for improvement~55°C, 60 s

↓No improvement~65°C, 45 s

*1 The number of cycles can be increased up to 45.
*2. 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 0.5 and 0.8 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 0.5 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.

[7] Application data

1. Detection of respiratory syncytial (RS) virus in clinical specimens
RNA of RS virus type A was detected from throat swab specimens using
THUNDERBIRD® Probe One-step qRT-PCR Kit with Applied Biosystems®
StepOnePlus™. The RNA (6.5 × 104 copies) purified by the QIAmp Virus RNA Kit(QIAGEN) was used in the assay after serial dilution from 100 to 10-5.
<Cycling conditions>
Step
Reverse transcription
Predenaturation
PCR
Denaturation(45 cycles)
Extension (annealing)

Temperature
50°C
95°C
95°C
60°C

Time
10 min
60 s
15 s
45 s

<Primer and probes>
Sequence

LengthGC

Tm

Forward primer

GCTCTTAGCAAAGTCAAGTTGAATGA

26-mer38.5%

65.4°C

Reverse primer

TGCTCCGTTGGATGGTGTATT[FAM]ACACTCAACAAAGATCAACTTCTGTCATCCAGC[TAMRA]

21-mer47.6%

66.3°C

33-mer42.4%

73.9°C

TaqMan® probe<Results>
The RS virus was successfully detected from the RNA purified from clinical specimens.
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2. Simultaneous quantification of multiple targets
HeLa S3 cells (4 × 105 cells in a six-well plate) were cultured for 20 h with or without
100 nM phorbol 12-myristate 13-acetate. Then, IL-1, TNF- and GAPDH genes weredetected from the HeLa total RNA (1–100 pg) using THUNDERBIRD® Probe One-step
qRT-PCR Kit. The genes were analyzed using singleplex and multiplex detection systems
with LightCycler® 96.
<Cycling conditions>
Step
Reverse transcription
Predenaturation
PCR
Denaturation(45 cycles)
Extension (annealing)

Temperature
50°C
95°C
95°C
60°C

Time
10 min
60 s
15 s
45 s

<Primers and probes>
IL-

Sequence

LengthGC

Forward primer

ACAGATGAAGTGCTCCTTCCA

21-mer

47.6%

63.7°C

Reverse primer

21-mer52.4%

64.3°C

TaqMan® probe

GTCGGAGATTCGTAGCTGGAT[FAM]CTCTGCCCTCTGGATGGCGG[TAMRA]

20-mer70.0%

73.5°C

TNF-

Sequence

LengthGC

Tm

Forward primer

CCCAGGGACCTCTCTCTAATC

21-mer57.1%

62.8°C

Tm

Reverse primer

ATGGGCTACAGGCTTGTCACT

21-mer52.4%

64.8°C

TaqMan® probe[Cy5]TGGCCCCAGGCAGTCAGATCATC

23-mer60.9%

75.3°C[BHQ2]GAPDH: human GAPD (GAPDH) endogenous control (VIC[R]/MGB probe, primer
limited) (Applied Biosystems, 4326317E)

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www.toyobo.co.jp/e/bio<Results>
The singleplex and multiplex detection systems showed similar results of PCR efficiency
and correlation coefficient (R2). The results indicated that this system facilitates analysisof the expression of multiple genes.

Multiplex
Singleplex

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IL-
Efficiency
R2
90.2%
0.989
91.6%
0.999

TNF-
Efficiency
R2
91.8%
0.999
92.9%
0.998

GAPDH
Efficiency
R2
89.6%
0.999
91.6%
0.999

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www.toyobo.co.jp/e/bio[8] Troubleshooting
Symptom

Cause
Incorrect setting of detector mode
for the fluorescence dye
Incorrect setting for data collection
Incorrect setting for sample position
Degradation of RNA
Inappropriate extension (annealing)
temperature or time

Low PCR efficiency,
low sensitivity,
divergence of results

Inappropriate concentration ofprimers or probes

Inappropriate design of primers orprobesInappropriate concentration of ROX
Reference dye
Failure or malfunction of device
Low quality of sample DNA

Absorption of RNA

Dispersion ofquantification

Excessive or insufficient template
RNA
Inappropriate extension (annealing)
temperature or time
Inappropriate concentration ofprimers or probes

Inappropriate design of primers orprobesDispersion of reaction volume

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Solution
Confirm the detector setting.
Confirm the data collection setting.
Reposition the sample tubes.
RNA should be purified again. RNA purification
method, reagents, and equipment should also bereconfirmed simultaneously.
Extension (annealing) temperature and timeshould be optimized according to the instructions(see [6] Optimization of conditions).
If the results are not improved by optimizing theextension (annealing) temperature and time,
primer or probe concentration should be
optimized according to the instructions (see [6]Optimization of conditions).
If the results are not improved by changing primer
and probe concentrations, the primer or probe
design should be reconfirmed (see [6]Optimization of conditions).
The concentration of ROX Reference dye should
be determined depending on the real-time cycler(see [5] Protocol).
Check the device.
Purify the RNA sample again by an appropriate
method. Contaminated genomic DNA may also
cause divergence of data by it being amplified. Insuch cases, primers and/or probes should beredesigned according to [3] Primer/probe design.
Alternatively, DNase I treatment of RNA is also
effective to improve this problem.
A small amount of RNA tends to be absorbed by
microtubes. Low-absorption tubes should be used.
Quantification should be performed in the linear
range of the standard curve.
Extension (annealing) temperature and timeshould be optimized according to the instructions(see [6] Optimization of conditions).
If the results are not improved by optimizing theextension (annealing) temperature and time,
primer or probe concentration should be
optimized according to the instructions (see [6]Optimization of conditions).
If the results are not improved by changing primer
and probe concentrations, the primer or probe
design should be reconfirmed (see [6]Optimization of conditions).
Insufficient reaction volume tends to reduce thesensitivity or reproducibility. The reaction volume
should be determined according to the instruction
manual of each real-time cycler.

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Symptom
Detection of signal in anegative control

Cause

SolutionContamination of positive samples
or PCR products

If the signal is detectable even when a fresh
negative control is used, all reagents should bechecked and prepared again.
TaqMan® probe itself may generate a nonspecificsignal by forming unexpected structures. In suchcases, the TaqMan® probe should be redesigned.

Design of TaqMan® probe is not
appropriate[9] Related products
Product nameHighly efficient cDNA synthesis kit for real-time PCR®

200 rxns

Code No.
QPS-201T
QPS-201
FSQ-101

Highly efficient cDNA synthesis master mix for real-time PCR®

200 rxns

FSQ-201

Highly efficient cDNA synthesis master mix for real-time PCR with genomic DNA

200 rxns

FSQ-301

One-step real-time PCR master mix for probe assay™

0.5 mL × 5

QRT-101

One-step real-time PCR master mix for SYBR® Green assay®

0.5 mL × 5

QRT-201

Highly efficient real-time PCR master mix®
®

Package
200 rxns

THUNDERBIRD SYBR qPCR Mix
ReverTra Ace qPCR RT Kit

ReverTra Ace qPCR RT Master Mix
remover

ReverTra Ace® qPCR RT Master Mix
with gDNA remover
RNA-direct Real-time PCR Master Mix
RNA-direct™ SYBR Green Real-time PCR Master Mix

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