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F0921K

Instruction manual ReverTra AceTM qPCR RT Kit 2004

ReverTra AceTM qPCR RT Kit

FSQ-101

200 reactions
Store at -20°CContents[1]Introduction[2]Components[3]Protocol
1. Template RNA for reverse transcription
2. Reverse transcription[4] Related protocol
1. DNase I treatment of total RNA[5] Troubleshooting[6] Related products

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.

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

1

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

Description
The ReverTra AceTM qPCR RT Kit is an efficient and convenient kit to synthesize highquality cDNAs for real-time PCR. This kit contains the highly efficient reverse
transcriptase “ReverTra AceTM” and a RT buffer optimized for the highly efficient synthesisof short-chain cDNAs suitable for real-time PCR. The protocol is simple, and the reaction
can be completed in 15 min.
ReverTra AceTM is a mutant-type M-MLV reverse transcriptase shows excellent efficiency.

Features-The optimized RT buffer and Primer Mix enable the highly efficient reverse
transcription from RNA to cDNA without additional requirements.
-The reaction can be completed in 15 min. The protocol does not contain an additional
RNase H treatment step to remove residual RNA after reverse transcription (PatentPending).
-Since the RT buffer is optimized for real-time PCR, the addition of 20% (v/v) of thesynthesized cDNA solution to the PCR solution does not inhibit the PCR reaction.
Therefore, this kit is suitable for the detection of low-expressing mRNAs.

[ 2 ] Components

The kit includes the following reagents which can be used for 200 applications of 10 μL
reactions. All reagents should be stored at -20 °C.
5 x RT Buffer
Enzyme Mix
Primer Mix
Nuclease-free water

400 μL100 μL100 μL
1000μL x 2

5 × RT BufferThis buffer contains MgCl2 and dNTPs.
Enzyme Mix
This solution contains the highly efficient reverse transcriptase “ReverTra AceTM” and an
RNase inhibitor.
Primer Mix
This solution contains random and Oligo (dT) primers. The primer ratio has been optimized
for the efficient reverse transcription of various RNA targets.
Nuclease-free waterThis nuclease-free water has been prepared without DEPC treatment.

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Tel (+86)-21-58794900

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

1

www.toyobo.co.jp/e/bio[ 3 ] Protocol

1.

Template RNA for reverse transcription

The following RNAs are appropriate for highly efficient reverse transcription.
(1)Total RNA
Total RNA usually contains 1-2% mRNA. Total RNA can be used as template directlywith this kit. RNA prepared by AGPC (Acid Guanidium-Phenol-Chloroform) or thecolumn method contains genomic DNA, so total RNA should be treated with DNase I
before transcription.
(2)Poly(A)+ RNA (mRNA)
Poly (A)+ RNA is useful 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.
2.

Reverse transcription(1)Denaturation of RNA
Incubate the RNA solution at 65 °C for 5 min, and keep on ice afterwards.
Notes-This step is important to increase the efficiency of the reverse transcriptase reaction.
-Do not add the other components at this step.
(2)Preparation of the reaction solution
Prepare the following reagents.
Nuclease-free water

X μL

5 x RT Buffer

2 μL

RT Enzyme Mix

0.5 μL

Primer Mix
RNATotal Volume0.5 μL
0.5 pg – 1 μg
10 μL

Notes
-The Primer Mix can be replaced by gene-specific primers. Gene-specific primers canbe used at a final concentration of 0.5 pmol/μL.
-This kit contains nuclease-free water for 200 reverse transcription reactions but not forthe dilution of RNA samples. Nuclease-free water prepared without DEPC-treatment isrecommended for dilution of RNA samples.

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Tel(81)-6-6348-3888
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FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

2www.toyobo.co.jp/e/bio(3) Incubate at 37 °C for 15 min.
(4) Heat at 98 °C for 5 min.
(5) Store the reacted solution* at 4 °C or – 20 °C*This solution can be used directly or after dilution for real-time PCR.

Notes
-The reaction time at 37 °C can be prolonged up to 1 hr.
-Up to 20% of the synthesized cDNA solution can be added to the PCR reaction solution.
-Reverse transcription with gene-specific primers tends to lead to non-specific PCRamplification. Maintaining the reaction temperature at 42-50 °C helps reduce nonspecific amplification.

JAPANTOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

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

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

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www.toyobo.co.jp/e/bio[ 4 ] 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 μL

Total RNA (<10 µg)

Y μL l10 x DNase I Buffer[e.g. 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 phenolVortex
Keep on ice for 5 min.
 Centrifuge at 12,000 rpm for 5 min.
Supernatant  Add 100 μL chloroform: isoamyl alcohol (24:1), Vortex
 Centrifuge at 12,000 rpm for 5 min. at 4 °C
Supernatant Add 100 μL 5 M ammonium acetate + 200 μL isopropanol+ [5 μL l 2 mg/mL glycogen* (for coprecipitation) : optional]
Vortex
Incubate at - 20 °C for 30 min.
 Centrifuge at 12,000 rpm for 10-15 min. at 4 °C
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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TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

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

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www.toyobo.co.jp/e/bio[ 5 ] Troubleshooting
Symptom
Low signal at real-time
PCR

Non-specific signal atreal-time PCR[ 6 ] Related products

Cause

Solution

Low purity of RNA

Repurify the RNA sample.

Degradation of RNA

Prepare the RNA sample again.

Excess or small amounts ofRNA

Optimal range of the RNA template for reverse
transcription is from 0.5 pg to 1 µg of RNA.

Inappropriate temperature
conditions

Perform the reaction according to this instruction
manual.

Excess amount of cDNA
solution compared to thetotal PCR reaction volume
Non-specific annealing ofgene specific primers

The maximum volume ratio of the cDNA solution to thePCR solution is 20% (v/v). Reduce the volume of thecDNA solution.
Change the reaction temperature from 37 °C to 42 50 °C.

Product nameHigh efficient revers transcriptaase
ReverTra AceTM

RNase inhibitor (Recombinant type)
Real-time PCR master mix for probe assay

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

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

RNA-direct™ Realtime PCR Master Mix

JAPAN
TOYOBO CO., LTD.
Tel(81)-6-6348-3888
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tech_osaka@toyobo.jp

Package
10,000 U

Code No.
TRT-1012,500 U
1 mL x 5

SIN-201
QPK-101

1 mL x 5

QPK-2011 mL x 5

QPK-2120.5 mL x 5

QRT-101

0.5 mL x 5

QRT-201CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

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

5