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F0930K

Instruction manual RNA-direct™ SYBR® Green Realtime PCR Master Mix 2004

RNA-direct ™

SYBR® Green Realtime PCR Master Mix
QRT-201

0.5mLx5

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

Introduction[2]

Components[3]

Primer design[4]Detection[5]

Specimens[6]

Protocol1.

Intercalation assay protocol using ABI PRISM® 7900HT

2.

Intercalation assay protocol using Roche LightCycler™

[7]

Related protocol
1. DNase I treatment of total RNA[8] Trouble shooting[9]

Related products[10] References

CAUTIONAll reagents in this kit are intended for research purposes. Do not use for diagnosis or clinical purposes. Please observe generallaboratory precaution and utilize safety 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.
-ABI PRISM® is a registered trademark of Perkin-Elmer Corporation.

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

Description

This product is a 2 × Master Mix for “1-step real-time PCR” using a thermostable DNApolymerase derived from Thermus thermophilus (Tth) HB81). Tth DNA polymerase
exhibits reverse transcriptase activity in the presence of Mn2+ ions. This system allows for“1-step real-time PCR”, including reverse transcription and PCR steps. This reagent canbe applied to an intercalation assay with SYBR® Green I.

Features-This reagent is suitable for high-throughput real-time PCR and increases reliability ofproduct, due to lowered risk of contamination.
-This reagent can be used in systems using glass capillaries (e.g., LightCycler, Roche
Molecular Systems, Inc.).
-This reagent can be used in systems using passive reference (e.g., ABI PRISM® 7700,
Applied Biosystems, Inc.). The passive reference dye does not affect any other systems.
-Hot Start technology, using anti-Tth DNA polymerase antibodies, allows for highspecificity and reproducible amplification.

[ 2 ] Components

This reagent includes the following components for 100 reactions, with a total of 50 μL perreaction. All reagents should be stored at -20 °C.

<QRT-201>
RNA-direct™ SYBR® Green Realtime PCR Master Mix 0.5mLx550mM Mg(OAc)20.5mLx5Notes:
This reagent can be stored at 4°C for up to 2 months, protected from light. For longer
storage, this reagent should be kept at -20°C and protected from light.

[ 3 ] Primer

1. Primer conditions

Primers should be designed according to the following guidelines:-Primer length: 20~30 mer-GC content of primer: 40~60%-Target length: ≤200 bp (optimally, 50~150 bp)
Notes:
Longer targets (>200 bp) reduce efficiency and specificity of amplification. The ideal
optimal target length range is 50~150 bp.

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www.toyobo.co.jp/bio[ 4 ] Detection

This reagent can be used with the following devices:

1. Normal devices, such as:-LineGene, Bioeretc.

2. Devices using a glass capillary or a passive reference, such as:-LightCycler, Roche Molecular Systems-ABI PRISM® 7000, 7700, and 7900, Applied Biosystems

ΔRnNotes: The passive reference mode of detectors should be set at “ROX”.

0102030Cycle40Fig. 1 Detection of β-actin expression with TaqMan® assay
Device: ABI PRISM® 7900HT
Target: β-actin
Assay method: TaqMan® probe
Template: cDNA from poly(A)+ RNA (HeLa cells)[ 5 ] Specimen

The following RNAs are appropriate for highly efficient 1-step real-time PCR.

1.

Total RNA
Total RNA typically contains 1~2% mRNA, which can be used as template directly withthis kit. RNA prepared by AGPC (Acid Guanidium-Phenol-Chloroform) or the column
method contains genomic DNA; therefore, the total RNA should be treated with DNase
I prior to transcription.

2. Poly(A)+ RNA (mRNA)
Poly(A)+ RNA can be used to detect low-level expressing mRNA. However, poly(A)+RNA should be treated carefully, because Poly(A)+ RNA is more sensitive to RNase thantotal RNA.

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www.toyobo.co.jp/bio[ 6 ] Protocol

1.

Intercalation assay protocol using ABI PRISM® 7900HT
The following is an intercalation assay protocol using ABI PRISM® 7900HT. If using
other detection devices, this protocol should be slightly altered, according to each
instruction manual.
(1) Preparation of reaction solutionComponent

VolumeFinal
Concentration

Nuclease-free water

15.5 μL
25 μL
2.5 μL1.0 μL1.0 μL5 μL

1x
2.5 mM
0.2 µM
0.2 µM

RNA-direct™ SYBR® Green Realtime PCR Master Mix

50mM Mn(OAc)2
10 pmol /μL (10 µM) Primer #1
10 pmol /μL (10 µM) Primer #2
Template RNA
Total RNA
Poly (A)+ RNA
Total volume<2.5 µg / 50 μL<500 ng / 50 μL

50 μL

Notes
-Primer concentrations can be further optimized, if needed. The optimal range of primers
is 0.2~0.6 µM. In the case of commercially available primers, those recommended
condition should be used.
-The final concentration of Mn(OAc)2 should be adjusted to 2~3.5 mM. Lower Mnconcentrations result in decreased non-specific amplification; higher Mn concentrations
result in increased amplification efficiency.
-Nuclease-free water prepared without DEPC-treatment is recommended.
(2) Cycling condition
The following is a cycling condition the “standard mode” of ABI PRISM® 7900HT.
<3-step cycle>
Denaturation :90 °C, 30 sec.

RT :61 °C, 20 min.

Pre-denaturation :95 °C, 1 min.

Denaturation :95 °C, 15 sec.

Annealing :55-65 °C, 15 sec.

Extension :74 °C, 45 sec. (data collection)

45 cycles

Melting curve analysis
Notes-The PCR Master Mix contains anti-Tth DNA polymerase antibodies for Hot Start PCR.
The first denaturation step (90 °C, 30 sec.) is sufficient to inactivate the antibodies. Do
not prolong this denaturation step.
-The annealing temperature should to Tm-5 °C. The optimal annealing temperature
range is 55-65°C.
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www.toyobo.co.jp/bio

2. Intercalation assay protocol using Roche LightCycler™
The following is an intercalation assay protocol using Roche LightCycler™. In the caseof other detection devices, this protocol should be slightly altered, according to each
instruction manual.
(1) Preparation of reaction solution
Component

VolumeFinal
Concentration

Nuclease-free water6.2 μL
10 μL
1 μL
0.4 μL0.4 μL
2 μL

1x2.5 mM
0.2 µM
0.2 µM

RNA-direct™ SYBR® Green Realtime PCR Master Mix

50 mM Mn(OAc)2
10 pmol /μL (10 µM) Primer #1
10 pmol /μL (10 µM) Primer #2Template RNA
Total RNA
Poly(A)+ RNA
Total volume<1 µ g / 20 μL<200 ng / 20 μL

20 μL

Notes
-The primer concentration can be further optimized, if needed. The optimal range ofprimers is 0.2~0.6 µM. In the case of commercially available primers, thoserecommended conditions should be used.
-The final concentration of Mn(OAc)2 should be adjust to 2~3.5 mM. Lower Mnconcentrations result in decreased non-specific amplification; higher Mn concentrations
result in increased the amplification efficiency.
-Nuclease-free water prepared without DEPC-treatment is recommended.
(2) Cycling condition

The following condition is recommended.
<2-step cycle>
Denaturation :90 °C, 30 sec.

RT :61 °C, 20 min.

Pre-denaturation :95 °C, 30 sec.

Denaturation :95 °C, 5 sec.

Annealing :55~65 °C, 10 sec.

Extension :74 °C, 15 sec. (data collection)

45 cycles

Notes-The PCR Master Mix contains anti-Tth DNA polymerase antibodies for Hot Start PCR.
The first denaturation step (90°C, 30 sec.) is sufficient to inactivate the antibodies. Do
not prolong this denaturation step.
-The temperature transition rate should be set to 20 °C/sec. Pool amplification may be
improved by adjusting the temperature transition rate to 2 °C/sec.
-If the target length is ≤ 200 bp, the extension time should be adjusted to 15 sec. Datacollection steps should be at least 15 sec.
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www.toyobo.co.jp/bio[7] Related Protocol

1.

DNase I treatment of total RNA

Total RNA prepared by general methods contains genomic DNA. Genomic DNA can beeliminated by the following method.
(1) Mix the following reagents.
Nuclease-free water

X μLTotal RNA (<10 µg)

Y μL

10 x DNase I Buffer[100 mM Tris-Cl, 20 mM MgCl2(pH 7.5)]

1 μL

RNase-free DNase I (10 U/μL)

0.5 μL

Total volume10 μL(2) Incubate on ice for 10~30 min.
(3) Purify the treated RNA according to the following step.
DNase I-treated RNA  Add nuclease-free water (adjust volume to 100 μL)
  Add 100 μL TE-saturated phenol
Vortex
Keep on ice for 5 min.
 Centrifuge at 12,000 rpm for 5 min.
Supernatant  Add 100 μL chloroform, Voltex
 Centrifuge at 12,000 rpm for 5 min.
Supernatant Add 5 μL 20mg/mL glycogen* (for coprecipitation) +100 μL 5 M ammonium
acetate + 200 μL isopropanol
Incubate at - 20 °C for 30 min.
 Centrifuge at 2,000 rpm for 5 min.
Discard supernatant
Precipitate Add 1 mL 70% ethanol Centrifuge at 12,000 rpm for 5 min.
Discard supernatant
Precipitate Dissolve in appropriate volume of nuclease-free water
RNA solution*Molecular biology grade
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www.toyobo.co.jp/bio[ 8 ] Troubleshooting
Symptom

No amplification

Variation in detection

Signals in blankreactions

Cause

Confirm the detector setting.

Incorrect setting for data collection.

Confirm the data collection setting.

Incorrect setting for sample position.

Reposition the sample tubes.

Inappropriate concentration ofprimers or probes.

Optimize primer or probe concentration according
to instructions (see [6]).

Inappropriate design of primers orprobes.

Confirm specificity and Tm of primers and probes.

Inappropriate cycle conditions.

Confirm Tm of the primers and probes.

Low purity or quality of samples

Check the purity or quality of sample DNA.

Low concentration of Mn(OAc)2.
Failure or malfunction of device

Increase the Mn concentration to 3.5 mM.
Check the device

Low quality of sample DNA.

Repurify DNA sample by phenol/chloroform
extraction and ethanol precipitation, or othermethod.
Optimize primer or probe concentration according
to the instructions (see [6]).

Inappropriate concentration ofprimers or probes.
Inappropriate design of primers orprobes.

Confirm specificity and Tm of primers and probes.

Inappropriate cycle conditions.

Confirm Tm of the primers and probes.

Variation of dispensed volume

Increase the reaction volume

Contamination
sample DNAs.

ofamplicons

Detection of a non-specificamplification.

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Solution

Incorrect setting of detector mode forthe fluorescence dye.

orUse fresh PCR grade water. Re-make primer
solution, probe solution, and master mix.
Optimize the primer and cycle conditions.

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

www.toyobo.co.jp/bio[ 9] Related products
Product nameHigh efficient cDNA synthesis kit for real-time PCR
ReverTra AceTM qPCR RT Kit
High efficient revers transcriptaase
ReverTra AceTM

RNase inhibitor (Recombinant type)
Realtime PCR master mix for probe assay

Realtime PCR Master Mix
Realtime PCR master mix for SYBR® Green assay
SYBR® Green Realtime PCR Master Mix
Realtime PCR master mix for SYBR® Green assay (improved version)
SYBR® Green Realtime PCR Master Mix –PlusOne-step realtime PCR master mix for probe assay

RNA-direct™ Realtime PCR Master Mix[10] References

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Package
200 rxns

Code No.
FSQ-101

10,000U

TRT-1012,500U
1mLx5

SIN-201
QPK-101

1mLx5

QPK-201

1mLx5

QPK-2120.5mLx5

QRT-1011) Myers T. W. and Gelfand D. H. , Biochemistry, 30: 7661-6 (1991)

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900
www.bio-toyobo.co.jp

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