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Instruction manual ReverTra Ace® qPCR RT Master Mix with gDNA remover1204

ReverTra Ace® qPCR RT Master Mix
with gDNA Remover
FSQ-301

200 reactions
Store at -20°C

Contents[1]

Introduction[2]

Components[3]

Protocol
1. RNA Template for reverse transcription
2. Reverse transcription[4]

Application data

[5]

Troubleshooting[6]

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.
- ReverTra Ace® is a registered trademark of Toyobo Co., Ltd., Japan.
JAPANTOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO Bio-Technology, CO., LTD.
Tel(86)-21-58794900.4140

FOR RESEARCH USE ONLY.

1NOT FOR HUMAN OR DIAGNOSTIC USE.

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

Introduction

Description
ReverTra Ace® qPCR RT Master Mix with gDNA remover is an efficient and convenient
kit, consisting of master mix reagents, to synthesize high quality cDNAs for real-time
PCR. The kit includes reagents for reverse transcription and for the removal of genomic
DNA [DNase I treatment].
In many cases, total RNA prepared using spin-columns or acid guanidium-phenolchloroform (AGPC) extraction methods contains small amount of genomic DNA. Any
contaminating genomic DNA will be amplified along with cDNA, especially whenprimer pairs are designed within the same exon or from pseudogenes. Amplification fromgenomic DNA can result in qualitative and quantitative inaccuracies.
The protocol consists of i) a genomic DNA degradation step using “gDNA remover” andii) a reverse transcription step. The two steps can be achieved sequentially without
purification or heat inactivation of DNase I.
ReverTra Ace® is a mutant M-MLV reverse transcriptase that shows excellent efficiency.

Features-“Genomic DNA degradation step” and “cDNA synthesis step” can be achieved
sequentially in approximately 30 min.
-The master mix reagents will not freeze at -20°C.
- Control, no reverse transcription experiments (no RT-Control) can be performed with
5x RT Master Mix II no-RT control.
-The master mix reagent contains random and oligo dT primers optimized for efficientreverse transcription.
-The reverse transcription reaction can be completed in 15 min. The protocol does notcontain an additional RNase H treatment step to remove residual RNA after reverse
transcription (Patent Pending).
-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 abundance mRNAs.

[2]

Components

The kit includes the following reagents, which can be used for 200 (FSQ-301) and 40(FSQ-301S) 10 µl reactions. All reagents should be stored at -20°C. For extended storage,-30°C is recommended.

gDNA Remover
4x DN Master Mix
5x RT Master Mix II
5x RT Maser Mix II no RT-Control
Nuclease-free water

FSQ-30110 l
440 l400 l40 l1000 l x 2

FSQ-301S (SAMPLE)
4 l
88 l
80 l
8 l
400 lgDNA remover“gDNA remover” is an optimized DNase I solution. 4x DN Master Mix and gDNA
remover should be mixed at a ratio of 50 : 1.

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4× DN Maser Mix“4x DN Master Mix” is a buffer solution that contains RNase inhibitor. Prior to use, a 1
in 50 volume of gDNA remover should be added to 4x DN Master Mix (e.g. 4x DN
Master Mix : gDNA remover = 440 l : 8.8 l or 4x DN Master Mix : gDNA remover =88 l : 1.8 l).
Notes
4x DN Master Mix with gDNA remover can be stored at -20°C for at least 3 months. The
mixture can be prepared in a smaller volume [e.g. 4x DN Master Mix: gDNA remover = 220 l :4.4 l].

5× RT Maser Mix IIThis reagent is a 5x master mix that contains highly efficient reverse transcriptase“ReverTra Ace®”, RNase inhibitor, oligo dT primer, random primer and dNTPs.
Notes
Be aware that “5x RT Master Mix II” and “5x RT Master Mix” in ReverTra Ace® qPCR RT
Master Mix (Code No. FSQ-201)” are not compatible.

5× RT Maser Mix II no-RT Control
The composition of “5x RT Master Mix II no-RT Control” is identical to that of “5x RT
Master Mix II” except that reverse transcriptase (RT) is omitted. This master mix can beused in a control experiment due to the absence of reverse transcriptase.
Nuclease-free waterThis nuclease-free water has been prepared without DEPC treatment.

[3]

ProtocolRNA template
Denaturation of RNA[Optional]

65°C, 5 min.
On iceGenomic DNA removal(DNase I treatment)

Nuclease-free Water
4x DN Master Mix (with “gDNA Remover”)
37°C, 5 min.

Reverse transcription(cDNA synthesis)

5xRT Master Mix II
37°C, 15 min.
(50°C, 5 min.) [Optional]
98°C, 5 min.
cDNA
qPCR

Flowchart of genomic DNA removal and cDNA synthesis
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FOR RESEARCH USE ONLY.

2NOT FOR HUMAN OR DIAGNOSTIC USE.

www.toyobo.co.jp/e/bio

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 directly as templatewith this kit.
(2)Poly(A)+ RNA (mRNA)
Poly(A)+ RNA is useful to detect low abundance mRNAs. However, poly(A)+ RNA
should be treated carefully because it is more sensitive to RNase than total RNA.

2.

Reverse transcription(1) Preparation of the “4x DN Master Mix” and “gDNA Remover” mixture.
Prior to use, a 1 in 50 volume of gDNA remover should be added to 4x DN MasterMix (e.g. 4x DN Master Mix : gDNA remover = 440 l : 8.8 l or 4x DN MasterMix : gDNA remover = 88 l : 1.8 l).
Notes
4x DN Master Mix with gDNA remover can be stored at -20°C for at least for 3 months. The
mixture can be prepared in a smaller volume [e.g. 4x DN Master Mix : gDNA remover = 220l : 4.4 l].

(2) Denaturation of RNA [optional]
Incubate the RNA solution at 65°C for 5 min, and then keep on ice.
Notes
- This step increases the efficiency of reverse transcription of RNA templates that form
secondary structures.
-This step should be performed before adding 4x DN Master Mix.
(3) Preparation of the DNase I reaction solution:
Prepare the following reagents on ice.
4x DN Master Mix
RNA template(4)

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2 l0.5 pg – 0.5g

Nuclease-free Water

X lTotal Volume

8 lIncubate at 37°C for 5 min.

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Preparation of the for reverse transcription solution;
Prepare the following reagents on ice.
Reacted solution from (4)8 l5x RT Master Mix II2 l

Total Volume

10 l

Notes
-The master mix reagent contains oligo dT and random primers. Do not use withspecific primers.
-For control experiments, “5x RT Master Mix II no RT-Control” should be used insteadof 5x RT Master Mix II. A control experiment without reverse transcription is usefulto prove whether amplicons originate from cDNA and/or genomic DNA.
-This kit contains nuclease-free water for 200 reverse transcription reactions. The kit
does not contain sufficient nuclease-free water for the dilution of RNA samples.
Nuclease-free water prepared without DEPC-treatment is recommended for thedilution of RNA samples.
-The reaction volume can be increased according to need.

(6)

Incubate at 37°C for 15 min(7)Incubate at 50°C for 5 min [optional](8)

Heat at 98°C for 5 min(9)Store the reacted solution* at 4°C or – 20°C

*This solution can be used directly or after dilution for real-time PCR.

Notes-“ReverTra Ace®” excels at high reaction temperatures (up to 50°C). This step may
increase the efficiency of the reverse transcription.
-Up to 20% of the synthesized cDNA solution can be added to the PCR reaction
solution.

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Tel(86)-21-58794900.4140

FOR RESEARCH USE ONLY.

4NOT FOR HUMAN OR DIAGNOSTIC USE.

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

Application data

(1) Efficiency of genomic DNA removal.
<Materials and Methods>
cDNA synthesis
Reagent: ReverTra Ace® qPCR RT Master Mix with gDNA remover(Code No.FSQ-301)
Template: HeLa total RNA 0.5 g /10 l reaction
Experiment conditions: The experiments were preformed with the following conditions.
4x DN Master Mix
5x RT Master Mix[DNase I treatment][Reverse transcription]
A
gDNA Remover (-)*
RTase (-)**
B
gDNA Remover (-)*
RTase (+)
CgDNA Remover (+)
RTase (-)**
DgDNA Remover (+)
RTase (+)
* 4xDN Master Mix without gDNA Remover**5x RT Master Mix II no-RT Control
Real-time PCR
Reagent: THUNDERBIRD® SYBR® qPCR Mix (Code No.QPS-201)
Template: cDNA 2 l/20 l reaction (cDNA solution: 10%)Target: -actin (188 bp)
Real-time cycler: Applied Biosystems 7900HT<Results>

DBA
C

No signal for the “C experiment” indicates that the contaminating genomic DNA in theRNA template was completely removed by “gDNA remover”.

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Tel(86)-21-58794900.4140

FOR RESEARCH USE ONLY.

5NOT FOR HUMAN OR DIAGNOSTIC USE.

www.toyobo.co.jp/e/bio(2) Comparison of cDNA yields<Materials and Methods>
cDNA synthesis
Reagents: -ReverTra Ace® qPCR RT Master Mix with gDNA remover(Code No.FSQ-301)-ReverTra Ace® qPCR RT Kit* (Code No. FSQ-101)*Previous version of the kit without gDNA remover.
Template:

HeLa total RNA 1 pg, 10 pg, 100 pg, 1 ng, 10 ng, 100 ng, 1 g/20 l reaction****Genomic DNA (100 ng) was added to the experiments using FSQ-301.

Real-time PCR
Reagent: THUNDERBIRD® SYBR® qPCR Mix (Code No.QPS-201)
Template: cDNA 2 l/20 l reaction (cDNA solution: 10%)Target: GAPDH (65 bp)
Real-time cycler: Applied Biosystems 7900HTCt<Results>

3634323028262422201816141210

y = -1.4846Ln(x) + 34.422
R2 = 0.999
FSQ-101
FSQ-301y = -1.4838Ln(x) + 34.038
R2 = 0.9991

1101001000

対数 (FSQ-101)
対数 (FSQ-301)

10000

1000001000000

RNA Quantity (pg)Despite the genomic DNA contamination, the results of ReverTra Ace® qPCR RT MasterMix with gDNA remover (Code No.FSQ-301) correlate highly with those of theReverTra Ace® qPCR RT Kit* (Code No. FSQ-101). Both reagents showed highly linear
standard curves in a broad concentration range.

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Tel(86)-21-58794900.4140

FOR RESEARCH USE ONLY.

6NOT FOR HUMAN OR DIAGNOSTIC USE.

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

Cause

Solution

Low purity of RNA

Repurify the RNA sample.

Degradation of RNA

Prepare fresh RNA sample. Diluted RNA templates havea tendency to degrade and to adsorb on the vessel walls.
RNA template for the reaction should be prepared from ahighly concentrated stock prior to use.
The recommended RNA concentration range for reverse
transcription is from 1 pg to 1 g in a 10 l reaction.
However, the optimal concentration of RNA templateshould be determined for each case.
The efficiency of reverse transcription of RNAs thatform secondary structures tends to be low. Incubation at65°C for 5 min and quenching prior to the reaction isusually effective on such templates. Also, the additional
step of 50°C for 5 min after the reaction at 37°C for 15min might be effective for such difficult templates.
Perform the reaction according to this instruction
manual.

Excess or small amount ofRNA

Secondary structure of RNA
template

Inappropriate temperature
conditions

Amplification in no-RT
control reaction

Excess amount of cDNA
solution compared to thetotal PCR reaction volume
Contamination of an excess
amount of genomic DNA inRNA template
Primer dimer formation

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Reduce the cDNA solution to less than 10%.

Repurify the RNA template. Contaminating genomicDNA of up to approximately 50 ng (per 10 l reaction)
can be treated. An excess amount of genomic DNA in
the RNA template can result in incomplete degradation
Optimize the PCR conditions or redesign the primers.
HPLC-grade primers sometimes improve PCR
specificity.

CHINA
TOYOBO Bio-Technology, CO., LTD.
Tel(86)-21-58794900.4140

FOR RESEARCH USE ONLY.

7NOT FOR HUMAN OR DIAGNOSTIC USE.

www.toyobo.co.jp/e/bio[ 6 ] Related products
Product namePackage
10,000 U

Code No.
TRT-101

RNase inhibitor (Recombinant type)2,500 U

SIN-201Real-time PCR master mix for probe assay
THUNDERBIRD® Probe qPCR Mix

1.67 mL x 3

QPS-101

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

1.67 mL x 3

QPS-201

High efficient cDNA synthesis kit for Real-time PCR
ReverTra Ace® qPCR RT Kit

200 reactions

FSQ-101

High efficient cDNA synthesis master mix for Real-time PCR
ReverTra Ace® qPCR RT Master Mix

200 reactions

FSQ-201

High efficient revers transcriptaase
ReverTra Ace®

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

CHINA
TOYOBO Bio-Technology, CO., LTD.
Tel(86)-21-58794900.4140

FOR RESEARCH USE ONLY.

8NOT FOR HUMAN OR DIAGNOSTIC USE.