www.toyobo.co.jp/e/bio
JAPAN CHINA
TOYOBO CO., LTD. TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel(81)-6-6348-3888 Tel (+86)-21-58794900
www.toyobo.co.jp/e/bio
FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.
1
Instruction manual ReverTra Ace
TM
qPCR RT Master Mix with gDNA remover2004 Ａ1172Ｋ
ReverTra Ace
TM
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
CAUTION
All 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.
www.toyobo.co.jp/e/bio
JAPAN CHINA
TOYOBO CO., LTD. TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel(81)-6-6348-3888 Tel (+86)-21-58794900
www.toyobo.co.jp/e/bio
FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.
1
[ 1 ] Introduction
[ 2 ] Components
Description
ReverTra Ace
TM
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-phenol-
chloroform (AGPC) extraction methods contains small amount of genomic DNA. Any
contaminating genomic DNA will be amplified along with cDNA, especially when primer
pairs are designed within the same exon or from pseudogenes. Amplification from genomic
DNA can result in qualitative and quantitative inaccuracies.
The protocol consists of i) a genomic DNA degradation step using “gDNA remover” and
ii) a reverse transcription step. The two steps can be achieved sequentially without
purification or heat inactivation of DNase I.
ReverTra Ace
TM
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 efficient
reverse transcription.
-The reverse transcription 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 (Patent Pending).
-Since the RT buffer is optimized for real-time PCR, the addition of 20% (v/v) of the
synthesized cDNA solution to the PCR solution does not inhibit the PCR reaction.
Therefore, this kit is suitable for the detection of low abundance mRNAs.
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.
FSQ-301
FSQ-301S
(SAMPLE)
gDNA Remover
10 μL
4 μL
4x RT Master Mix
440 μL
88 μL
5x RT Master Mix II
400 μL
80 μL
5x RT Maser Mix II no RT-Control
40 μL
8 μL
Nuclease-free water
1000μL x 2
400 μL
gDNA 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.
www.toyobo.co.jp/e/bio
JAPAN CHINA
TOYOBO CO., LTD. TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel(81)-6-6348-3888 Tel (+86)-21-58794900
www.toyobo.co.jp/e/bio
FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.
2
[ 3 ] Protocol
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 μL : 8.8 μL or 4x DN Master Mix : gDNA remover = 88 μL :
1.8 μL).
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 µl : 4.4
μL].
5× RT Maser Mix II
This reagent is a 5x master mix that contains highly efficient reverse transcriptase
“ReverTra Ace
TM”
, 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
TM
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 be
used in a control experiment due to the absence of reverse transcriptase.
Nuclease-free water
This nuclease-free water has been prepared without DEPC treatment.
Flowchart of genomic DNA removal and cDNA synthesis
RNA template
Denaturation of RNA 65°C, 5 min.
[Optional] On ice
Genomic DNA removal Nuclease-free Water
(DNase I treatment) 4x DN Master Mix (with “gDNA Remover”)
37°C, 5 min.
Reverse transcription 5xRT Master Mix II
(cDNA synthesis) 37°C, 15 min.
(50°C, 5 min.) [Optional]
98°C, 5 min.
cDNA
qPCR
www.toyobo.co.jp/e/bio
JAPAN CHINA
TOYOBO CO., LTD. TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel(81)-6-6348-3888 Tel (+86)-21-58794900
www.toyobo.co.jp/e/bio
FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.
3
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 template
with 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 Master Mix
(e.g. 4x DN Master Mix : gDNA remover = 440 μL : 8.8 μL or 4x DN Master Mix :
gDNA remover = 88 μL : 1.8 μL).
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
μL :
4.4
μL].
(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.
(4) Incubate at 37°C for 5 min.
4x DN Master Mix
2 μL
RNA template
0.5 pg – 0.5
µ
g
Nuclease-free Water
X μL
Total Volume 8 μL
www.toyobo.co.jp/e/bio
JAPAN CHINA
TOYOBO CO., LTD. TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel(81)-6-6348-3888 Tel (+86)-21-58794900
www.toyobo.co.jp/e/bio
FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.
4
(5) Preparation of the for reverse transcription solution;
Prepare the following reagents on ice.
Notes
-The master mix reagent contains oligo dT and random primers. Do not use with specific
primers.
-For control experiments, “5x RT Master Mix II no RT-Control” should be used instead
of 5x RT Master Mix II. A control experiment without reverse transcription is useful to
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 the dilution
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
TM
” 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.
Reacted solution from (4)
8 μL
5x RT Master Mix II
2 μL
Total Volume
10 μL
www.toyobo.co.jp/e/bio
JAPAN CHINA
TOYOBO CO., LTD. TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel(81)-6-6348-3888 Tel (+86)-21-58794900
www.toyobo.co.jp/e/bio
FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.
5
[ 4 ] Application data
(1) Efficiency of genomic DNA removal.
<Materials and Methods>
cDNA synthesis
Reagent: ReverTra Ace
TM
qPCR RT Master Mix with gDNA remover
(Code No.FSQ-301)
Template: HeLa total RNA 0.5 µg /10 μL reaction
Experiment conditions: The experiments were preformed with the following conditions.
4x DN Master Mix
[DNase I treatment]
5x RT Master Mix
[Reverse transcription]
A
gDNA Remover (-)*
RTase (-)**
B
gDNA Remover (-)*
RTase (+)
C
gDNA Remover (+)
RTase (-)**
D
gDNA Remover (+)
RTase (+)
* 4xDN Master Mix without gDNA Remover
**5x RT Master Mix II no-RT Control
Real-time PCR
Reagent: THUNDERBIRD
TM
SYBR
®
qPCR Mix (Code No.QPS-201)
Template: cDNA 2 μL /20 μL reaction (cDNA solution: 10%)
Target: β-actin (188 bp)
Real-time cycler: Applied Biosystems 7900HT
<Results>
No signal for the “C experiment” indicates that the contaminating genomic DNA in the
RNA template was completely removed by “gDNA remover”.
B
D
A
C
www.toyobo.co.jp/e/bio
JAPAN CHINA
TOYOBO CO., LTD. TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel(81)-6-6348-3888 Tel (+86)-21-58794900
www.toyobo.co.jp/e/bio
FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.
6
(2) Comparison of cDNA yields
<Materials and Methods>
cDNA synthesis
Reagents: -ReverTra Ace
TM
qPCR RT Master Mix with gDNA remover
(Code No.FSQ-301)
-ReverTra Ace
TM
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 µg
/20 μL reaction
**
**Genomic DNA (100 ng) was added to the experiments using FSQ-301.
Real-time PCR
Reagent: THUNDERBIRD
TM
SYBR
®
qPCR Mix (Code No.QPS-201)
Template: cDNA 2μL /20 μL l reaction (cDNA solution: 10%)
Target: GAPDH (65 bp)
Real-time cycler: Applied Biosystems 7900HT
<Results>
Despite the genomic DNA contamination, the results of ReverTra Ace
TM
qPCR RT Master
Mix with gDNA remover (Code No.FSQ-301) correlate highly with those of the ReverTra
Ace
TM
qPCR RT Kit
*
(Code No. FSQ-101). Both reagents showed highly linear standard
curves in a broad concentration range.
y = -1.4838Ln(x) + 34.038
R
2
= 0.9991
y = -1.4846Ln(x) + 34.422
R
2
= 0.999
10
12
14
16
18
20
22
24
26
28
30
32
34
36
1
10
100 1000
10000 100000
1000000
RNA Quantity (pg)
Ct
FSQ-101
FSQ-301
対数
(FSQ-101)
対数 (FSQ-301)
www.toyobo.co.jp/e/bio
JAPAN CHINA
TOYOBO CO., LTD. TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel(81)-6-6348-3888 Tel (+86)-21-58794900
www.toyobo.co.jp/e/bio
FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.
7
[ 5 ] Troubleshooting
Symptom Cause Solution
Low signal after real-
time PCR
Low purity of RNA
Repurify the RNA sample.
Degradation of RNA
Prepare fresh RNA sample. Diluted RNA templates have
a tendency to degrade and to adsorb on the vessel walls.
RNA template for the reaction should be prepared from a
highly concentrated stock prior to use.
Excess or small amount of
RNA
The recommended RNA concentration range for reverse
transcription is from 1 pg to 1 µg in a 10μL reaction.
However, the optimal concentration of RNA template
should be determined for each case.
Secondary structure of RNA
template
The efficiency of reverse transcription of RNAs that
form secondary structures tends to be low. Incubation at
65
°C for 5 min. and quenching prior to the reaction is
usually effective on such templates. Also, the additional
step of 50°C for 5 min. after the reaction at 37°C
for 15
min. might be effective for such difficult templates.
Inappropriate temperature
conditions
Perform the reaction according to this instruction
manual.
Excess amount of cDNA
solution compared to the
total PCR reaction volume
Reduce the cDNA solution to less than 10%.
Amplification in no-RT
control reaction
Contamination of an excess
amount of genomic DNA in
RNA template
Repurify the RNA template. Contaminating genomic
DNA of up to approximately 50 ng (per 10μL reaction)
can be treated. An excess amount of genomic DNA in
the RNA template can result in incomplete degradation
Primer dimer formation
Optimize the PCR conditions or redesign the primers.
HPLC-grade primers sometimes improve PCR
specificity.
www.toyobo.co.jp/e/bio
JAPAN CHINA
TOYOBO CO., LTD. TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel(81)-6-6348-3888 Tel (+86)-21-58794900
www.toyobo.co.jp/e/bio
FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.
8
[ 6 ] Related products
Product name
Package
Code No.
High efficient revers transcriptaase
ReverTra Ace
TM
10,000 U
TRT-101
RNase inhibitor (Recombinant type)
2,500 U
SIN-201
Real-time PCR master mix for probe assay
THUNDERBIRD
TM
Probe qPCR Mix
1.67 mL x 3
QPS-101
Real-time PCR master mix for SYBR
®
Green assay
THUNDERBIRD
TM
SYBR
®
qPCR Mix
1.67 mL x 3
QPS-201
High efficient cDNA synthesis kit for Real-time PCR
ReverTra Ace
TM
qPCR RT Kit
200 reactions
FSQ-101
High efficient cDNA synthesis master mix for Real-time PCR
ReverTra Ace
TM
qPCR RT Master Mix
200 reactions
FSQ-201
