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F1267K

SuperPrep Cell Lysis & RT Kit for qPCR 2004

SuperPrep Cell Lysis & RT Kit for qPCR
TM

SCQ-101

SuperPrep Cell Lysis Kit for qPCR

100 reactions
Store at -20°C

TM

SCQ-201

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
SuperPrepTM Cell Lysis & RT Kit for qPCR (Code No. SCQ-101) consists of “Lysis
Reagents” and “RT Reagents” for synthesis of cDNA templates for real-time PCR assays.
“Lysis Reagents” prepares cell lysates containing RNAs that can be used as templates forreverse transcription. “RT Reagents” contains 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.
SuperPrepTM Cell Lysis Kit for qPCR (Code No. SCQ-201) is an option of “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 2 h. High-quality cDNA can be synthesized using
highly efficient reverse transcriptase “ReverTra AceTM” 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
Adherent/
Cell Name

Species

Remarks

Non-adherent
1

A431

Adherent

H. sapiens

epidermoid carcinoma cell line

2

C2C12

Adherent

M. musculus

myoblast cell line3

Caco-2Adherent

H. sapiens

colon adenocarcinoma cell line

4

CHO-K1

Adherent

C. griseus

ovary cell line

5

COLO205

Non-adherent

H. sapiens

colon adenocarcinoma cell line

6

DLD-1

Adherent

H. sapiens

colon adenocarcinoma cell line7

HCT-15Adherent

H. sapiens

colon adenocarcinoma cell line

8

HDF

Adherent

H. sapiens

primary foreskin fibroblasts (primary cell)

9

HEK293

Adherent

H. sapiens

embryonic kidney cell line

10

HeLa S3

Adherent

H. sapiens

cervix carcinoma cell line

11

HepG2

Adherent

H. sapiens

hepatocellular carcinoma cell line

12

HUVEC

Adherent

H. sapiens

umbilical vein endothelial cells (primary cell)

13

JurkatNon-adherent

H. sapiens

T lymphocyte cell line

14

K562

Non-adherent

H. sapiens

myelogenous leukemia cell line

15

KUSA-A1

Adherent

M. musculus

bone marrow stromal stem cell line

16

L929

Adherent

M. musculus

aneuploid fibrosarcoma cell line

17

MCF7

Adherent

H. sapiens

breast adenocarcinoma cell line

18

Neuro2aAdherent

M. musculus

neuroblastoma cell line

19

NIH-3T3

Adherent

M. musculus

embryo fibroblast cell line

20

PC12

Adherent

R. norvegicus

adrenal pheochromocytoma cell line

21

rMSC

Adherent

R. norvegicus

bone marrow stromal stem cells (primary cell)

22

THP-1

Non-adherent

H. sapiens

acute monocytic leukemia cell line

23

U937

Non-adherent

H. sapiens

leukemic monocyte lymphoma cell line

-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. The DNase I reaction can be stopped by adding “Stop
Solution”. Additional purification or heating steps are not necessary.
-Various real-time PCR reagents can be applied
The synthesized cDNA can be used in various real-time PCR assay (TaqMan®
probe, SYBR® Green etc). The examples using THUNDERBIRDTM qPCR Mix(Code No. QPS-101, QPS-201) are detailed in [4] 1.
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.

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www.toyobo.co.jp/e/bio[ 2 ] Components

The kits include the following reagents, which can be used for 100 (SCQ-101), 20 (SCQ101S), 100 (SCQ-201) and 20 (SCQ-201S) reactions. SCQ-101 and SCQ-101S containtwo separate packages named “Lysis Reagents” and “RT Reagents”, respectively. All
reagents should be stored at -20°C.
SuperPrepTM Cell Lysis & RT Kit for qPCR (Code No. SCQ-101, SCQ-101S)<Lysis Reagents>
Lysis Solution*1
gDNA Remover
Stop Solution
RNase Inhibitor

SCQ-101
5.5 mL33 μL
1.1 mL
55μL

SCQ-101S (SAMPLE)
1.1 mL6.6 μL220 μL11μL<RT Reagents *2>
5 × RT Master Mix
5 × RT Master Mix no-RT Control
Nuclease-free Water

860 μL86 μL
1.7 mL × 2

172 μL
17 μL
680 μL

SuperPrerTM Cell Lysis Kit for qPCR (Code No. SCQ-201, SCQ-201S)

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

SCQ-201
5.5 mL33 μL
1.1 mL
55 μL

SCQ-201S (SAMPLE)
1.1 mL6.6 μL220 μL
11 μL

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

Lysis Solution
Lysis Solution should be used with gDNA Remover to protect RNA in the cell lysate. The
solution also minimizes the inhibition of cDNA synthesis by cell components.
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.
Stop Solution
Stop Solution contains components that inhibit enzymes in the cell lysate.
RNase Inhibitor
RNase Inhibitor should be used with Stop 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 AceTM”, 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]
Add 0.3 μL gDNA Remover to 49.7 μL Lysis Solution. The mixture can beprepared as one solution according to sample numbers. When using a
multi-channel pipet, the amount of the mixture should be increased byabout 10% because of waste.
Notes
Do not store the mixed solution.

g.

Add 50 μL Lysis Solution with gDNA Remover 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 lysate can be stored on ice up to 2 h.

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[Preparation of Stop Solution with RNase Inhibitor]
Add 0.5 μL RNase Inhibitor to 9.5 μL Stop Solution. The mixture can beprepared as one solution according to sample numbers. When using a
multi-channel pipet, the amount of the mixture should be increased about
10% because of waste.

j.

Add 10 μL Stop Solution with RNase Inhibitor to wells.

k.

Mix the plate gently using a plate mixer or tapping with hands for 30 sec
and incubate at room temperature for 1.5 min.
Notes
In the case of cells that exhibit high RNase activity, the lysate should be
incubated on ice to obtain better reproducibility.

l.

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 2 h. The lysate should be stored at -80°C for 2 weeks.
-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.

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

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.

(3) For cells cultured in plates (except 96-well plates)a.

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.: SCQ101) 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) l.] 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.
50°C, 5 min.
98°C, 5 min.
4°C, Hold(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) THUNDERBIRDTM Probe qPCR Mix (Code No. QPS-101) for TaqMan® assay
The detailed conditions should be referred to in the instruction manual of
THUNDERBIRDTM Probe qPCR Mix (Code No. QPS-101).
a. Reaction setup

Reagent
DW

Reaction volume
50 μL
20 μL
X μL
X μL

Final
concentration

THUNDERBIRDTM 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 THUNDERBIRDTM 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 sec.
Maximum(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 sec.
Capillary cycler (e.g. Roche LightCycler™ 1.x, 2.0)
30 sec.
General real-time cyclers (e.g. Applied Biosystems 7700,
60 sec.
7500, 7900HT [normal block], StepOne™, StepOnePlus™
Agilent cyclers, BioFlux cyclers)*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 sec.
7500, 7900HT [normal block], StepOne™, StepOnePlus™
Agilent cyclers, BioFlux cyclers)

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www.toyobo.co.jp/e/bio(2) THUNDERBIRDTM SYBR® qPCR Mix (Code No. QPS-201) for SYBR® Green I
assay
The detailed conditions should be referred to in the instruction manual of
THUNDERBIRDTM SYBR® qPCR Mix (Code No. QPS-201).
a. Reaction setup

Reagent
DW

Reaction volume
50 μL
20 μL
X μL
X μL

THUNDERBIRDTM 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~7.5 μL
50 μL

*1

Final
concentration

~3μL
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 and probe conditions described in the instruction
manual of THUNDERBIRDTM SYBR® qPCR Mix (Code No. QPS-201).
<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 sec.
Maximum(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 sec.
Capillary cycler (e.g. Roche LightCycler™ 1.x, 2.0)
30 sec.
General real-time cyclers (e.g. Applied Biosystems 7700,
60 sec.
7500, 7900HT [normal block], StepOne™, StepOnePlus™
Agilent cyclers, BioFlux cyclers)*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 sec.
7500, 7900HT [normal block], StepOne™, StepOnePlus™
Agilent cyclers, BioFlux cyclers)*3 Detailed conditions for the melting curve analysis should be referred to in the instruction

manuals of the real-time PCR reagents.

(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) l) 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 μL

b. 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 sec.
Maximum
55°C
20 sec.
Maximum
95°C
60 sec.
Maximum
95°C
15 sec.
Maximum
60°C
60 sec.
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40 cycles11www.toyobo.co.jp/e/bio(2) 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

Reagent
DW

Reaction volume
50 μL
20 μL
X μL
X μL

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 μL

b. 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).
<Three-step cycle>
Pre-denaturation:Reverse transcription:
Denaturation:
Annealing
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Temperature
Time
Ramp
90°C
30 sec.
Maximum
55°C
20 sec.
Maximum
95°C
60 sec.
Maximum
55°C
15 sec.
Maximum
74°C
60 sec.
Maximum(data collection should be set at the extension step)
Melting / Dissociation Curve Analysis

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40 cycles12www.toyobo.co.jp/e/bio[ 5 ] Application data

1. Comparison of the efficiency with purified RNA
SuperPrepTM Cell Lysis & RT Kit for qPCR (Code No SCQ-101) synthesized cDNA using
the lysate from 2.5 × 104 HEK293 cells in a 40 μL reaction. cDNA was synthesized using
66.6 ng of total RNA, corresponding to 2.5 × 104 HEK293 cells, using a cDNA synthesis
kit (ReverTra AceTM 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 SuperPrepTM
facilitated the expression analysis by real-time PCR without time-consuming RNA
purification steps.

Purified RNA (Ct)

R2 = 0.983

SuperP rep ® Lysate (Ct)

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 THUNDERBIRDTM
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 SuperPrepTM 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|
Company A

35

35

30

30

25

25

Ct

Ct

SuperPrepTM

20

20
15

15
0

5

10

15

20

0

5

10

IL-1β

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1520

well no.

well no.

IL-6β-actin

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www.toyobo.co.jp/e/bio

IL-6

ΔCt (IL-6-β-actin)

Ct (IL-6)

SuperPrepTM

PMA

Mean

Mean

S.D.

ΔΔCt

Z'

(+)

26.96

6.27

0.14

-3.85

0.62

-3.88

0.59

(-)

30.24

10.12

0.35

(+)

26.50

4.30

0.34

(-)

29.44

8.18

0.19

Company A

IL-1β

ΔCt (IL-1β-β-actin)

Ct (IL-1β)

SuperPrepTM

Company A

Mean

S.D.

ΔΔCt

Z'

28.62

7.93

0.15

-5.94

0.74

33.99

13.87

0.38
-6.19

0.61

PMA

Mean

(+)
(-)
(+)

28.00

5.80

0.47

(-)

33.26

11.99

0.34

3. Stability test of cell lysates
cDNA were synthesized from lysates that had been left on ice for 0–24 h after lysing of
4×104 HeLa and U937 cells using SuperPrepTM. β-actin genes were detected using
TaqMan® real-time PCR assay with THUNDERBIRDTM 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 2 h. Lysates fromU937 cells showed higher RNase activity than the other cells and tended to deteriorate instorage over 2 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.
U937

25

40

20

35

15

30

Ct

Ct

HeLa

10

15

0
0

10
on ice (hr)Company A

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25
20

5

0

20

10
on ice (hr)20SuperPrep™

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www.toyobo.co.jp/e/bio[ 6 ] Troubleshooting
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 2 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 mixing Lysis Solution and Stop
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.

Real-time PCR master mix for probe assay
THUNDERBIRDTM Probe qPCR Mix

1.67 mL × 3

QPS-101

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

1.67 mL × 3

QPS-201

0.5 mL × 5

QRT-101

0.5 mL × 5

QRT-201

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

RNA-direct™ Real-time PCR Master Mix®

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

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Tel (+86)-21-58794900

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15