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1
Instruction manual ReverTra Ace
TM
qPCR RT Master Mix 2004 Ｆ1173Ｋ
ReverTra Ace
TM
qPCR RT Master Mix
FSQ-201 200 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] Related protocol
1.
DNase I treatment of total RNA
[6] Troubleshooting
[7] 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.
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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 is an efficient and convenient reagent to synthesize
high quality cDNAs for real-time PCR. The master mix reagent (5x) contains the highly
efficient reverse transcriptase “ReverTra Ace
TM
”, primers and buffer optimized for highly
efficient synthesis of short-chain cDNAs suitable for real-time PCR. The protocol is simple,
and the reaction can be completed in 15 min.
ReverTra Ace
TM
is a mutant M-MLV reverse transcriptase that shows excellent efficiency.
Features
-5x Master Mix reagent contains all components for reverse transcription.
The Master Mix reagent will not freeze at -20°C.
-No reverse transcription control experiments (no RT-Control) can be performed.
-The master mix reagent contains random and oligo dT primers optimized for efficient
reverse transcription.
-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 (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-201) and 40
(FSQ-201S) 10 µl reactions. All reagents should be stored at -20°C. For extended storage,
-30°C is recommended.
FSQ-201
FSQ-201S
(SAMPLE)
5x RT Master Mix
400 μL
80 μL
5x RT Maser Mix no RT-Control
40 μL
8 μL
Nuclease-free water
1000 μL x 2
400 μL
5× RT Maser Mix
This reagent is a 5x master mix that contains highly efficient reverse transcriptase
“ReverTra Ace
TM”
, RNase inhibitor, oligo dT primer, random primer, MgCl
2
and dNTPs .
Notes
Be aware that “5x RT Master Mix” and “5x RT Master Mix II” in ReverTra Ace
TM
qPCR RT
Master Mix with gDNA remover (Code No. FSQ-301) are not compatible.
5× RT Maser Mix no-RT Control
The composition of “5x RT Master Mix no-RT Control” is identical to that of “5x RT
Master Mix” except that the reverse transcriptase (RT) is omitted. This master mix can be
used in control experiments due to the absence of reverse transcriptase.
Nuclease-free water
This nuclease-free water has been prepared without DEPC treatment.
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2
[ 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 directly as template
with this kit. RNA prepared using acid guanidium-phenol-chloroform (AGPC) or the
spin-column 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 abundance mRNAs. However, poly(A)
+
RNA
should be treated carefully because it is more sensitive to RNase than total RNA.
2. Reverse transcription
(1) 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 5x RT Master Mix.
(2) Preparation of the reaction 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 no RT-Control” should be used instead of
5x RT Master Mix. A control experiment without reverse transcription is useful to prove
whether amplicons originate from cDNA and/or genomic DNA.
-The reaction volume can be increased according to need.
-Master mix reagents should be spun-down prior to use due to high viscosity.
5x RT Master Mix
2 μL
RNA template
1 pg – 1
µ
g
Nuclease-free Water
X μL
Total Volume 10 μL
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3
-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.
(3) Incubate at 37°C for 15 min.
(4) Incubate at 50°C for 5 min. [optional]
(5) Heat to 98°C for 5 min.
(6) 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.
-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.
www.toyobo.co.jp/e/bio
JAPAN CHINA
TOYOBO CO., LTD. TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
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FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.
4
[ 4 ] Application data
<Materials and Methods>
cDNA synthesis
Reagent: ReverTra Ace
TM
qPCR RT Master Mix (Code No.FSQ-201)
Template: HeLa total RNA 2 pg-2 µg /20 μL reaction
Real-time PCR
Reagent: THUNDERBIRD
TM
SYBR
®
qPCR Mix (Code No.QPS-201)
Template: cDNA 2 μL /20 μL reaction (cDNA solution: 10%)
Targets: Typical house-keeping genes
Real-time cycler: Applied Biosystems 7900HT
<Results>
Template
RNA (pg)
Log
(RNA
amount)
Ct of qPCR
ATP5F TFRC RPLP1 RPLP2 RPS18
2
0.301
33.76
31.16
32.89
32.54
20
1.301
31.43
30.73
27.70
30.05
28.65
200
2.301
28.64
27.29
24.44
26.72
25.22
2,000
3.301
25.41
23.79
21.12
23.31
21.98
20,000
4.301
21.86
20.43
17.69
19.88
18.42
200,000
5.301
18.65
17.09
14.14
16.59
15.10
1,000,000
6.000
16.03
15.03
11.63
14.37
13.09
2,000,000
6.301
15.42
14.28
11.11
13.53
12.28
/20μl
Slope
-3.280
-3.303
-3.384
-3.284
-3.368
R2
0.999
0.999
1.000
1.000
0.999
Eff.
101.8%
100.8%
97.5%
101.6%
98.1%
High linearity and no crossing over of the standard curves of five housekeeping genes
suggest that the reagent shows high performance in a broad concentration range.
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5
[ 5 ] Related Protocol
1. DNase I treatment of total RNA
Total RNA prepared by general methods contains genomic DNA. Genomic DNA can be
eliminated by the following method.
(1) Mix the following reagents.
Nuclease-free water X μL
Total RNA (<10
µ
g)
Y μL
10 x DNase I Buffer
[e.g. 100 mM Tris-Cl, 20 mM MgCl
2
(pH 7.5)]
1 μL
RNase-free DNase I (10 U/μL) 0.5 μL
Total volume
10 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: 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 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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FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.
6
[ 6 ] 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 10 μL 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 genomic
DNA in RNA template
Redesign the primers to prevent amplification from
genomic DNA. Or treat the template RNA with DNase I
prior to reverse transcription.
Primer dimer formation
Optimize the PCR conditions or redesign the primers.
HPLC-grade primers sometimes improve PCR
specificity.
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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
[ 7 ] 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 with gDNA remover
ReverTra Ace
TM
qPCR RT Master Mix
with gDNA remover
200 reactions
FSQ-301
