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F1675K

SuperPrep II Cell Lysis & RT Kit for qPCR 2004

SuperPrep TM II Cell Lysis & RT Kit for qPCR
SCQ-401

SuperPrep TM II Cell Lysis Kit for qPCR

SCQ-501

100 reactions
Store at -20°C

100 preparations
Store at -20°C

Contents[1]

Introduction[2]

Components[3]

Protocol1. Preparation of cell lysate
2. Reverse transcription[4]

Real-time PCR
1. For assays using two-step real-time PCR reagents
2. For assays using one-step real-time PCR reagents[5]

Application data

[6]

Troubleshooting[7]

Related products

CAUTIONAll reagents in this kit are intended for research purposes. Do not use for diagnostic or clinical purposes. Please observe
general laboratory safety precautions while using this kit.

-LightCycler™ is a trademark of Idaho Technology, Inc. and Roche Molecular Systems, Inc.
-SYBR® is a registered trademark of Molecular Probes, Inc.
-TaqMan® is a registered trademark of Roche Molecular Systems, Inc.

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

Description
SuperPrep™ II Cell Lysis & RT Kit for qPCR (Code No. SCQ-401) consists of “Lysis
Reagents” and “RT Reagents” for synthesis of cDNA templates for real-time PCR assays.
“Lysis Reagents” prepare cell lysates containing RNAs that can be used as templates forreverse transcription. “RT Reagents” contain reagents for reverse transcription, optimized
for efficient cDNA synthesis from crude lysates. The synthesized cDNA can be applied toreal-time PCR directly. This assay system is suitable for high-throughput assays.
SuperPrep™ II can directly move to the cDNA synthesis step after adding lysis solution tocells, whereas the previous version requires Stop Solution after the Lysis step. Moreover,SuperPrep™ II realizes stable analysis in a wider range of mamalian cells, including
primary cells. With this assay system, it is possible to synthesize template cDNA for realtime PCR from cultured cells conveniently and quickly.
SuperPrep™ II Cell Lysis Kit for qPCR (Code No. SCQ-501) is an option for “Lysis
Reagents”. The cell lysate prepared by “Lysis Reagents” can be applied to one-step realtime PCR.

Features-RNA purification is not necessary.
Cell lysates can be prepared as effective templates for cDNA synthesis, reducing thetotal assay time.
-High-quality cDNA can be obtained from cell lysates.
The optimized lysis solution efficiently inhibits RNA degradation during treatment. RNAin the lysate is stable on ice for at least 6 h. High-quality cDNA can be synthesized using
highly efficient reverse transcriptase “ReverTra Ace™” with low contamination of
genomic DNA because of preceding DNase I treatment. The reverse transcriptase issupplied as a master mix reagent containing optimally mixed primers (random and oligodT) to achieve effective cDNA synthesis.

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Table 1 Cells tested by this system
Cell

Adherent/

Name

Non-adherent

1

HPA

2

HEK

3

Species

Remarks

Adherent

H. sapiens

preadipocytes (primary cell)

Adherent

H. sapiens

epidermal keratinocytes (primary cell)

HA

Adherent

H. sapiens

astrocytes (primary cell)

4

HDF

Adherent

H. sapiens

dermal fibroblasts (primary cell)

5

HFLS

Adherent

H. sapiens

fibroblast-like synoviocytes (primary cell)

6

HBEpC

Adherent

H. sapiens

bronchial epithelial cells (primary cell)

7

HUVEC

Adherent

H. sapiens

umbilical vein endothelial cells (primary cell)

8

HPAEC

Adherent

H. sapiens

pulmonary artery endothelial cells (primary cell)

9HC

Adherent

H. sapiens

chondrocytes (primary cell)

10

HOb

Adherent

H. sapiens

osteoblasts (primary cell)

11

HSkMC

Adherent

H. sapiens

skeletal muscle cells (primary cell)

12

HAOSMC

Adherent

H. sapiensaortic smooth muscle cells (primary cell)

13

HFDPC

Adherent

H. sapiens

hair follicle dermal papilla cells (primary cell)

14

HMSC

Adherent

H. sapiens

marrow stromal cell (primary cell)

15

A431

Adherent

H. sapiens

epidermoid carcinoma cell line

16

HeLa S3

Adherent

H. sapiens

cervix carcinoma cell line

17

HepG2

Adherent

H. sapiens

hepatocellular carcinoma cell line

18

C2C12

Adherent

M. musculus

myoblast cell line

19

NIH-3T3

Adherent

M. musculus

embryo fibroblast cell line

20

MDCK

Adherent

C. familiaris

kidney cell line

21

CHO-K1

Adherent

C. griseus

ovary cell line

22

JurkatNon-adherent

H. sapiens

T lymphocyte cell line

23

K562

Non-adherent

H. sapiens

myelogenous leukemia cell line

24

THP-1

Non-adherent

H. sapiens

acute monocytic leukemia cell line

25

U937

Non-adherent

H. sapiens

leukemic monocyte lymphoma cell line

26

HMNC

Non-adherent

H. sapiensmononuclear cells (primary cell)-Reduction of dispersion on high-throughput assay
Decreasing the small-volume dispensing and dilution steps from the protocol
reduced the dispersion of data in a high-throughput assay. Integrating oromitting some steps improved the usability. Cells can be lysed on the culture
plate without pipetting, and DNase I treatment can be performed at the sametime as the lysis step. Additional stop solution, purification or heating steps
are unnecessary.
-Various real-time PCR reagents can be applied
The synthesized cDNA can be used in various real-time PCR assays(TaqMan® probe, SYBR® Green etc). The examples using THUNDERBIRD™
qPCR Mix (Code No. QPS-101, QPS-201) and KOD SYBR™ qPCR Mix (CodeNo. QKD-201) are detailed in [4] 1.
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In addition, the cell lysate can be applied to one-step real-time PCR reagents.
The examples using RNA-direct™ real-time PCR Master Mix (Code No. QRT101, 201) are detailed in [4] 2.

[ 2 ] Components

The kits include the following reagents, which can be used for 100 (SCQ-401), 20 (SCQ401S) and 100 (SCQ-501) reactions. SCQ-401 and SCQ-401S contain two separate
packages named “Lysis Reagents” and “RT Reagents”, respectively. All reagents should be
stored at -20°C.
SuperPrep™ II Cell Lysis & RT Kit for qPCR (Code No. SCQ-401, SCQ-401S)<Lysis Reagents>
Lysis Solution*1
gDNA Remover
RNase Inhibitor

SCQ-401
6.5 mL33 μL
110 μLSCQ-401S (SAMPLE)
1.3 mL6.6 μL
22 μL

860 μL86 μL
1.7 mL × 2

172 μL
17 μL
680 μL<RT Reagents*2>
5 × RT Master Mix
5 × RT Master Mix no-RT Control
Nuclease-free Water

SuperPrep™ II Cell Lysis Kit for qPCR (Code No. SCQ-501)

Lysis Solution*1
gDNA Remover
RNase Inhibitor*1*2

SCQ-501
6.5 mL33 μL
110 μL

Lysis Solution can be stored at 2–8°C for 5 months. However, Lysis Solution contains
gDNA Remover and RNase Inhibitor, and should not be stored.
For extended storage of “RT Reagents”, -30°C is recommended.

Lysis Solution
Lysis Solution should be used with gDNA Remover and RNase Inhibitor to protect RNA
in the cell lysate. The solution also minimizes the inhibition of cDNA synthesis by cellcomponents.
gDNA Remover
gDNA remover is an optimized DNase I solution to degrade contaminated gDNAs in thecell lysate. gDNA Remover should be used with Lysis Solution.
RNase Inhibitor
RNase Inhibitor should be used with Lysis Solution to inhibit RNase in the cell lysate.

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5× RT Maser Mix
This reagent is a 5× master mix that contains highly efficient reverse transcriptase“ReverTra Ace™”, RNase Inhibitor, oligo dT primer, random primer and dNTPs.
5× RT Maser Mix no-RT Control
The composition of “5× RT Master Mix no-RT Control” is identical to that of “5× RT
Master Mix,” except that the reverse transcriptase (RT) is omitted. This master mix can beused in control experiments because of the absence of reverse transcriptase.
Nuclease-free Water
This nuclease-free water has been prepared without DEPC treatment.

[ 3 ] Protocol

1.

Preparation of cell lysates(1) For adherent cells cultured in a 96-well culture platea.

Seed an appropriate number of cells in a 96-well culture plate.
Notes
Lysis Solution can treat 1×101–7×104 cells. However, excessive cells
cause an insufficient lysis, inhibition of RT-PCR and partial digestion of
genomic DNA. The cell number for treatment should be determined bya preliminary experiment when treating >104 cells.

b.

Culture and incubate the cells.

c.

Remove the medium from the wells.

d.

Add 100 μL of PBS(-) to the wells.

e.

Remove PBS(-) from the wells.

f.

[Preparation of Lysis Solution with gDNA Remover and RNase Inhibitor ]Add 0.3 μL gDNA Remover and 1 μL RNase Inhibitor to 58.7 μL Lysis
Solution. The mixture can be prepared as one solution according tosample numbers. When using a multi-channel pipet, the amount of the
mixture should be increased by about 10% because of waste.
Notes
Do not store the mixed solution.

g.

Add 60 μL Lysis Solution with gDNA Remover and RNase Inhibitor to wells.

h.

Mix the plate gently using a plate mixer or tapping with hands for 30 sec
and incubate at room temperature for 4.5 min.
Notes
The incubation time can be increased up to 10 min. The lysate can be
stored on ice up to 6 h.

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Transfer the plate to ice.
Notes
-cDNA should be synthesized using the lysate as soon as possible,
although the stability of the RNA in the lysate has been confirmed as atleast 6 h. The lysate should be stored at -80°C for 2 months.
-The synthesized cDNA can be stored stably.

(2) For non-adherent cells cultured in a 96-well culture platea.

Seed an appropriate number of cells in a 96-well culture plate.
Notes
Lysis Solution can treat 1×101–7×104 cells. However, excessive cells
cause an insufficient lysis, inhibition of RT-PCR and partial digestion of
genomic DNA. The cell number for treatment should be determined bya preliminary experiment when treating >104 cells. The cell number forthe assay should be adjusted by removing an appropriate number ofcells from wells.

b.

Culture and incubate the cells.

c.

Centrifuge the plate at 2,000 rpm for 5 min using a plate centrifuge.

d.

Remove the medium.
Notes
The medium should be removed by pipetting or aspiration. Decantation
is not recommended.

e.

Add 100 μL of PBS(-).
Notes
Pipetting is not necessary.

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f.

Centrifuge the plate at 2,000 rpm for 5 min using a plate centrifuge.

g.

Remove the PBS(-).

h.

Go to [3] 1.(1) f.

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www.toyobo.co.jp/e/bio(3) For cells cultured in plates (except 96-well plates)
If using anything other than a 96-well plate, refer to Table 1 below for thenumber of cells to seed per well and the volumes of PBS(-) and Lysis Solution
to add per well.
Table 1

Cell Number and Buffer Volumes for Different Plate Formats

Plate format

Number of cells toseed per well

Volume of PBS(-)
to add per well384-well plate

4x103 cells

100μl

Volume of Lysis
Solution*1 to add per
well
16μl

48-well plate2x104 cells

250μl

120μl

24-well plate

4x104 cells

500μl

240μl

12-well plate8x104 cells

1000μl480μl

6-well plate

1x105 cells

2000μl

960μl*1 Includes gDNA Remover and RNase Inhibitor.

(4) For harvested cellsa.

Harvest the cells into a centrifuging tube and count the cell number.

b.

Pellet the cells and suspend the cells in PBS(-).

c.

Adjust the cell density to < 1×107 cells/mL by PBS(-).

d.

Dispense 5 μL of cell suspension into the wells.

e.

Go to [3] 1.(1) f.

2.

Reverse transcription(1) [Preparation of reaction mixture (40 μL reaction)]
Add 8 μL 5×RT Master Mix to 24 μL Nuclease-free water in a tube.
The mixture can be prepared as one solution according to sample numbers.
When using a multi-channel pipet, the amount of the mixture should be
increased by about 10% because of waste.
Notes
For the negative control experiments without the reverse transcriptase,
5× RT Master Mix no-RT Control can be used. The kit (Code No.: SCQ401) contains sufficient no-RT control for 10 reactions.
(2) Dispense 32 μL reaction mixtures into a PCR plate.
(3) Add 8 μL of the lysate [from [3] 1.(1) i.] and mix gently.
(4) Spin down the solutions.
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www.toyobo.co.jp/e/bio(5) Incubate the mixture as follows:
37°C, 15 min*1
50°C, 5 min
98°C, 5 min
4°C, Hold*1 The incubation time can be increased up to 60 min.

(6) Go to the real-time PCR step.
Notes
The synthesized cDNA can be stored at 4°C for a week and -20°C longterm.

[ 4 ] Real-time PCR
1.

For assays using two-step real-time PCR reagentsThe synthesized cDNA (from [3]2,(6)) can be applied to various kinds of real-time PCR
assay directly. Ten to 15% (v/v) cDNA can be added to the PCR reaction to achieve good
reproducibility. The optimal amount of cDNA should be determined by preliminary tests.
The conditions for several real-time PCR reagents are shown below as examples.
(1) THUNDERBIRD™ Probe qPCR Mix (Code No. QPS-101) for TaqMan® assay
The detailed conditions should be referred to in the instruction manual of
THUNDERBIRD™ Probe qPCR Mix (Code No. QPS-101).
a. Reaction setup

Reagent
DW

Reaction volume
50 µl
20 µl
X µl
X µl

Final
concentration

THUNDERBIRD™ Probe qPCR Mix

25 µl

10 μl

1×

Forward primer
Reverse primer

15 pmol
15 pmol

6 pmol6 pmol

0.3 μM
0.3 μM

TaqMan® Probe

10 pmol

4 pmol

0.2 μM*1

50× ROX reference dye

1 μl / 0.1 μl 0.4 μl / 0.04 μl 1× / 0.1×*2

cDNA solution
Total~7.5 µl
50 µl

~3 µl
20 µl

*1 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.
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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, BioFlux cyclers, etc.

Optimal dye concentration(dilution ratio)
1× (50:1)
0.1× (500:1)
Not requiredb. PCR cycling condition
The following table shows the recommended thermal conditions using primers designed
according to the recommended primer and probe conditions described in the instruction
manual of THUNDERBIRD™ Probe qPCR Mix (Code No. QPS-101).
<Two-step cycle>
Pre-denaturation:
Denaturation:
Extension:

Temperature

Time

Ramp

95°C

60 sec*1

Maximum

15 sec*2

95°C
Maximum
60°C
60 secMaximum(data collection should be set at the extension step)

40 cycles*1 In the anti-Taq antibody hot start PCR system, the pre-denaturation can be completed

within 60 sec. The pre-denaturation time should be determined according to therecommendations of each real-time cycler. If the optimal pre-denaturation time cannot
be determined, the time should be set at 60 sec.
Table 2 The recommended pre-denaturation time for each real-time cycler
Real-time cycler
Pre-denaturation timeHigh-speed cycler (e.g. Applied Biosystems 7500Fast)
20 secCapillary cycler (e.g. Roche LightCycler™ 1.x, 2.0)
30 secGeneral real-time cyclers (e.g. Applied Biosystems 7700,
60 sec7500, 7900HT [normal block], StepOne™, StepOnePlus™
Agilent cyclers, BioFlux cyclers, Bio-Rad CFX, QIAGEN
Rotor-Gene)*2 The following table shows the optimal denaturation times for each real-time cycler. If

the optimal denaturation time cannot be determined, the time should be set at 15 sec.
Table 3 The recommended denaturation time for each real-time cycler
Real-time cycler
Denaturation timeHigh-speed cycler (e.g. Applied Biosystems 7500Fast)
3 sec
Capillary cycler (e.g. Roche LightCycler™ 1.x, 2.0)
5 sec
General real-time cyclers (e.g. Applied Biosystems 7700,
15 sec7500, 7900HT [normal block], StepOne™, StepOnePlus™
Agilent cyclers, BioFlux cyclers, Bio-Rad CFX, QIAGEN
Rotor-Gene)JAPANTOYOBO CO., LTD.
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www.toyobo.co.jp/e/bio(2) KOD SYBR® qPCR Mix (Code No. QKD-201) for SYBR® Green I assay
The detailed conditions should be referred to in the instruction manual of KOD SYBR®
qPCR Mix (Code No. QKD-201).
a. Reaction setup

Reagent
DW

Reaction volume
50 µl
20 µl
X µl
X µl

KOD SYBR® qPCR Mix

25 µl

10 μl

1×

Forward primer
Reverse primer

10 pmol
10 pmol

4 pmol4 pmol

0.2 μM
0.2 μM

50× ROX reference dye

1 μl / 0.1 μl 0.4 μl / 0.04 μl 1× / 0.1×*1

cDNA solution
Total~7.5 µl
50 µl

*1

Final
concentration

~3 µl
20 µl

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, BioFlux cyclers, etc.

Optimal dye concentration(dilution ratio)
1× (50:1)
0.1× (500:1)
Not requiredb. PCR cycling conditions
The following table shows the recommended thermal conditions using primers designed
according to the recommended primer conditions.
<Two-step cycle>
Pre-denaturation:
Denaturation:
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Temperature

Time

Ramp

95°C

60 sec*1

Maximum

15 sec*2

95°C
Maximum
60°C
60 secMaximum(data collection should be set at the extension step)
Melting / Dissociation Curve Analysis*3

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40 cycles9www.toyobo.co.jp/e/bio*1 In the anti-Taq antibody hot start PCR system, the pre-denaturation can be completed

within 60 sec. The pre-denaturation time should be determined according to therecommendations of each real-time cycler. If the optimal pre-denaturation time cannot
be determined, the time should be set at 60 sec.
Table 2 The recommended pre-denaturation time for each real-time cycler
Real-time cycler
Pre-denaturation timeHigh-speed cycler (e.g. Applied Biosystems 7500Fast)
20 secCapillary cycler (e.g. Roche LightCycler™ 1.x, 2.0)
30 secGeneral real-time cyclers (e.g. Applied Biosystems 7700,
60 sec7500, 7900HT [normal block], StepOne™, StepOnePlus™
Agilent cyclers, BioFlux cyclers, Bio-Rad CFX, QIAGEN
Rotor-Gene)*2 The following table shows the optimal denaturation times for each real-time cycler. If

the optimal denaturation time cannot be determined, the time should be set at 15 sec.
Table 3 The recommended denaturation time for each real-time cycler
Real-time cycler
Denaturation timeHigh-speed cycler (e.g. Applied Biosystems 7500Fast)
3 sec
Capillary cycler (e.g. Roche LightCycler™ 1.x, 2.0)
5 sec
General real-time cyclers (e.g. Applied Biosystems 7700,
15 sec7500, 7900HT [normal block], StepOne™, StepOnePlus™
Agilent cyclers, BioFlux cyclers, Bio-Rad CFX, QIAGEN
Rotor-Gene)*3 Detailed conditions for the melting curve analysis should be referred to in the instruction

manuals of the real-time PCR reagents.
(3) THUNDERBIRD™ SYBR® qPCR Mix (Code No. QPS-201) for SYBR® Green I
assay
The detailed conditions should be referred to in the instruction manual of
THUNDERBIRD™ SYBR® qPCR Mix (Code No. QPS-201).
a. Reaction setup

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Reagent
DW

Reaction volume
50 µl
20 µl
X µl
X µl

Final
concentration

THUNDERBIRD™ SYBR® qPCR Mix

25 µl

10 μl

1×

Forward primer
Reverse primer

15 pmol
15 pmol

6 pmol6 pmol

0.3 μM
0.3 μM

50× ROX reference dye

1 μl / 0.1 μl 0.4 μl / 0.04 μl 1× / 0.1×*1

cDNA solution
Total

~5 µl
50 µl

~2 µl
20 µl

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See [4]1(2) Table 1.

b. PCR cycling conditions
See [4]1(2) b.
(3) Other real-time PCR reagentsThe synthesized cDNA (from [3]2(6)) can be applied to various kinds of real-time PCR
assay directly. Ten to 15% (v/v) cDNA can be added to the PCR reaction to achieve good
reproducibility. The optimal amount of cDNA should be determined by preliminary tests.
The detailed conditions should be set for each real-time PCR reagent.
Notes
In the case of real-time PCR reagents with hot start technology based on chemical
modification, the cDNA should be added up to 10% (v/v) in the PCR reaction.

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2.

For assays using one-step real-time PCR reagentsThe cell lysates (from [3]1(1) i) can be applied to various kinds of one-step real-time PCR
assay directly. The optimal amount of cDNA should be determined by preliminary tests.
The conditions for several one-step real-time PCR reagents are shown below as an example.
(1) RNA-direct™ Real-time PCR Master Mix (Code No. QRT-101) for TaqMan®
assay.
The detailed conditions should be referred to in the instruction manual of RNA-direct™
Real-time PCR Master Mix (Code No. QRT-101).
a. Reaction setup

Reagent
DW

Reaction volume
50 µl
20 µl
X µl
X µl

Final
concentration

RNA-direct™ Real-time PCR Master Mi

25 µl

10 μl

1×

50 mM Mn(OAc)2

2.5 µl1 µl

2.5 mM

Forward primer
Reverse primer

15 pmol
15 pmol

6 pmol6 pmol

0.3 μM
0.3 μM

TaqMan® Probe

10 pmol

4 pmol

0.2 μM

Cell lysate ([3]1(1) l)Total

2.5–5 µl
50 µl

1–2 µl20 µlb. PCR cycling condition
The following table shows the recommended thermal conditions using primers designed
according to the recommended primer and probe conditions described in the instruction
manual of RNA-direct™ Real-time PCR Master Mix (Code No. QRT-101) for TaqMan®
assay.
<Two-step cycle>
Pre-denaturation:Reverse transcription:
Denaturation:
Denaturation
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Temperature
Time
Ramp
90°C
30 secMaximum
55°C
20 secMaximum
95°C
60 secMaximum
95°C
15 secMaximum
60°C
60 secMaximum(data collection should be set at the extension step)CHINA
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Probe One-step qRT-PCR Kit (Code No. QRZ-101) for TaqMan® assay.
The detailed conditions should be referred to in the instruction manual of
THUNDERBIRD™ Probe One-step qRT-PCR Kit (Code No. QRZ-101).
a. Reaction setup
Reaction volume

Final

50 µl

20 µl

RNase free water
2× Reaction buffer
DNA Polymerase
RT Enzyme Mix

X µl
25 µl
1.25 µl1.25 µl

X µl
10 μl
0.5 μl
0.5 μlForward primer
Reverse primer
TaqMan® probe

25 pmol
25 pmol
10 pmol

10 pmol
10 pmol
4 pmol

0.5 μM
0.5 μM
0.2 μM

1μl / 0.1 μl

0.4 μl / 0.04 μl

1× / 0.1×*1

1 unit

0.4 units

2.5~5 µl

1~2 µl

50 µl

20 µl

50× ROX Reference dye(Uracil-N-glycosylase)*2 [optional]
Cell lysate ([3]1(1) l)

Concentration
1×

1 unit / 50 µl*1 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.
*2 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.
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 requiredb. PCR cycling condition
The following table shows the recommended thermal conditions using primers designed
according to the recommended primer and probe conditions described in the instruction
manual of THUNDERBIRD™ Probe One-step qRT-PCR Kit (Code No. QRZ-101) for
TaqMan® assay.
(2) THUNDERBIRD™
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www.toyobo.co.jp/e/bio(UNG treatment)
Reverse transcription
Pre-denaturation
Denaturation
Extension (annealing)(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.

(3) RNA-direct™ SYBR® Green Real-time PCR Master Mix (Code No. QRT-201) for
SYBR® Green I assay.
The detailed conditions should be referred to in the instruction manual of RNA-direct™
SYBR® Green Real-time PCR Master Mix (Code No. QRT-201).
a. Reaction setup
Reaction volume
50 µl
20 µl
X µl
X µl

Reagent
DW

Final
concentration

RNA-direct™ SYBR® Green
Real-time PCR Master Mix

25 µl

10 μl

1×

50 mM Mn(OAc)2

2.5 µl1 µl

2.5 mM

Forward primer
Reverse primer

10 pmol
10 pmol

4 pmol4 pmol

0.2 μM
0.2 μM

Cell lysate ([3]1(1) l)Total

2.5–5 µl
50 µl

1–2 µl20 µlb. PCR cycling condition
The following table shows the recommended thermal conditions using primers designed
according to the recommended primer conditions described in the instruction manual of
RNA-direct™ SYBR® Green Real-time PCR Master Mix (Code No. QRT-201).

TemperatureJAPAN
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Time

Ramp

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Pre-denaturation:Reverse transcription:
Denaturation:
Annealing
Extension:<Three-step cycle>
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Temperature

Time

90°C
30 secMaximum
55°C
20 secMaximum
95°C
60 secMaximum
55°C
15 secMaximum
74°C
60 secMaximum(data collection should be set at the extension step)
Melting / Dissociation Curve Analysis

40 cycles

Ramp

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www.toyobo.co.jp/e/bio[ 5 ] Application data

1. Comparison of the efficiency with purified RNA
SuperPrep™ II Cell Lysis & RT Kit for qPCR (Code No SCQ-401) synthesized cDNA
using the lysate from 2.5 × 104 HUVEC in a 40 μL reaction. cDNA was synthesized using
66.6 ng of total RNA, corresponding to 2.5 × 104 HUVEC, using a cDNA synthesis kit(ReverTra Ace™ qPCR RT Master Mix [Code No. FSQ-201]) in a 40 μL reaction. Fifteen
housekeeping genes were then analyzed by SYBR® Green real-time PCR using thesynthesized cDNA.
High correlation was observed between the two methods. The method using SuperPrep™
II facilitated the expression analysis by real-time PCR without time-consuming RNA
purification steps.

2. Evaluation of the assay variation
HeLa S3 cells were incubated with or without 100 nM phorbol 12-myristate 13-acetate(PMA) for 24 h after seeding at 2 ×104 cells/well in a 96-well culture plate. cDNA weresynthesized from the lysates prepared from the cells washed with PBS(-).IL-6, IL-1β and
β-actin genes were detected by TaqMan® real-time PCR assay with THUNDERBIRD™
Probe qPCR Mix (Code No. QPS-101). After compensation of the Cts of IL-6 and IL-1β bythat of β-actin, the ∆∆Ct between with or without PMA and Z’ factors*1 were calculated.
Z’ factors from SuperPrep™ II were superior to that from the other system (Company A).
*1 The Z’ factor is a simple statistical parameter that is used to assess the quality of high-throughput screening (HTS) assays. A Z’ score of 0.5 is generally considered to indicate
good quality Z’ can be calculated by the following formula.
Z’= 1-3 x [∆ Ct(+) standard deviation + ∆ Ct(-) standard deviation]/| ∆ ∆ Ct|
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Company A

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ΔCt (IL-6-β-actin)

Ct (IL-6)

IL-6
PMA

Mean

Mean

S.D.

ΔΔCt

Z'

(+)

25.35

3.45

0.20

-4.05

0.66

(-)

29.60

7.50

0.26

(+)

25.64

2.24

0.19

-3.94

0.64

(-)

29.76

6.18

0.29

SuperPrep™ II

Company A

IL-1β

ΔCt (IL-1β-β-actin)

Ct (IL-1β)
PMA

Mean

Mean

S.D.

ΔΔCt

Z'

(+)

27.83

5.93

0.18

-6.81

0.66

(-)

34.85

12.75

0.59

(+)

28.42

5.01

0.28

-7.38

0.59

(-)

35.98

12.39

0.74

SuperPrep™ II

Company A

3. Stability test of cell lysates
cDNA were synthesized from lysates that had been left on ice for 0–6 h after lysing of
4×104 HeLa and U937 cells using SuperPrep™ II. β-actin genes were detected using
TaqMan® real-time PCR assay with THUNDERBIRD™ Probe qPCR Mix (Code No. QPS101). The results were compared with that from the other company’s system (Company A).
The results suggest that the RNA in the cell lysates is stable for at least 6 h.
Notes
RNase activity depends on the type and number of cells. The cell lysates should beplaced on ice after preparation and cDNA should be synthesized immediately after
preparing the lysates to minimize RNA degradation.
HeLa S3

U937[ 6 ] Troubleshooting

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Symptom

Cause

Solution

No or low signal on
real-time PCR

Excessive number of cells
on lysate preparation

Excessive cellular compounds may inhibit cDNA
synthesis and qPCR steps. Decrease the number of cells
in the lysate preparation or dilute the cell lysates by thesame solution before cDNA synthesis.
-cDNA should be synthesized immediately after
preparing the cell lysates, especially on the cells
exhibiting high RNase activity.
(Cell lysates from general cells can be stored stably on
ice for at least 6 h.)-The cell lysates should be stored at -80°C avoiding
freezing and thawing.
-Harvested cells should be stored at -80°C after washing
PBS(-).
Ten to 15% (v/v) cDNA solution can be added to the PCR
reaction. However, tolerance of the amount of the cDNA
solution in the reaction may vary. The optimal amount ofcDNA should be determined by preliminary tests. The
conditions for several real-time PCR reagents are shown
below as examples.
Mix thoroughly after adding Lysis Solution.

Degradation of RNA

Excess amount of cDNA
solution compared with thetotal PCR reaction volume

Low yield

Cell lysis step wasinsufficient[ 7 ] Related products

Product name

Package

Code No.

THUNDERBIRD™ Probe qPCR Mix

1.67 mL × 3

QPS-101

Real-time PCR master mix for SYBR® Green assay
KOD SYBR® qPCR Mix

1.67 mL × 3

QKD-201

Real-time PCR master mix for SYBR® Green assay
THUNDERBIRD™ SYBR® qPCR Mix

1.67 mL × 3

QPS-201

1.25 mL × 2

QRZ-101

RNA-direct™ Real-time PCR Master Mix

0.5 mL × 5

QRT-101

One-step Real-time PCR master mix for SYBR® Green assay
RNA-direct™ SYBR® Green Realtime PCR Master Mix

0.5 mL × 5

QRT-201

Real-time PCR master mix for probe assay

One-step Real-time PCR master mix for probe assay

THUNDERBIRD™ Probe One-step qRT-PCR Kit
One-step Real-time PCR master mix for probe assay

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NOTICE TO PURCHASER:

LIMITED LICENSE

The use of this product is for research use only and is covered by a license from QIAGEN Group. No rights to use this product to perform oroffer any diagnostic and/or companion diagnostic services are granted by the supply of this product expressly, by implication or by estoppel.
Should you wish to use this product for any other purpose not covered by this license, please contact QIAGEN Corporate Business
Development at bd@qiagen.com.

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TOYOBO CO., LTD.
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CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.