1.
Quant Reverse
Transcriptase
For first-strand cDNA synthesis and
two-step RT-PCR
www.tiangen.com
RT080530
Quant Reverse Transcriptase
Cat. no. ER103
Kit Contents
Contents
ER103-03
50 rxns
ER103-04
100 rxns
Quant Reverse
Transcriptase
50 μl
2 × 50 μl
10× RT Buffer
150 μl
2 × 150 μl
Handbook
1
1
Storage
Quant Reverse Transcriptase should be shipped on dry ice and
stored immediately upon receipt at –20°C.
Introduction
Quant Reverse Transcriptase is a new, unique enzyme, different
from the reverse transcriptases of Moloney Murine Leukemia Virus
(MMLV) or Avian Myeloblastosis Virus (AMV). Quant Reverse
Transcriptase is a recombinant heterodimeric enzyme expressed in
E. coli. It has a high affinity for RNA, which enables efficient and
sensitive reverse transcription of RNA templates with high GC
content or complex secondary structures, leading to high yields of
cDNA.
Application
RT-PCR, Real-Time PCR, 3’ and 5’ RACE PCR, prime extension, cDNA
library construction, serial analysis of gene expression (SAGE), etc.
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Important Notes
1.
The protocol is optimized for use with 50 ng to 2 μg of RNA.
With >2 μg RNA, scale up the reaction linearly to the
appropriate volume.
2.
Set up all reactions on ice to minimize the risk of RNA
degradation.
3.
Separate denaturation and annealing steps are generally not
necessary. However, for some RNAs with a high degree of
secondary structure, a denaturation step may be desired. If so,
denature the RNA in RNase-free water before reaction setup:
incubate the RNA for 5 min at 65°C, then place immediately on
ice. Do not denature the RNA in the reaction mix.
4.
When using oligo-dT primers, a primer length of at least 12
nucleotides and a final concentration of 1 μM is recommended.
5.
Operate carefully to minimize RNA degradation and cross
contamination.
Starting Template
Reverse transcriptases are used in vitro for first-strand cDNA
synthesis with RNA as the starting template. The efficiency of the
reaction is highly dependent on the quality and quantity of the
starting RNA template.
1.
It is important to have intact RNA as starting template. Even
trace amounts of contaminating RNases in the RNA sample
can cause RNA cleavage, resulting in shortened cDNA products.
2.
Chemical impurities, such as protein, poly-anions (e.g.,
heparin), salts, EDTA, ethanol, phenol, and other solvents, can
affect the activity and processivity of the reverse ranscriptase.
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Protocol
The protocol is optimized for use with 50 ng to 2 μg of RNA.
With >2 μg RNA, scale up the reaction linearly to the appropriate
volume.
1.
Thaw template RNA on ice. Thaw the primer solutions, 10x RT
Buffer (supplied), Super pure dNTP, and RNase-free ddH2O at
room temperature (15–25°C). Place on ice immediately after
thawing. Mix each solution by vortexing, and centrifuge briefly
to collect residual liquid from the sides of the tubes.
2.
Dilute the RNA inhibitor to final concentration of 10 U/μl in 1x
RT Buffer which has been pre-cooled in ice. Mix thoroughly
and carefully by vortexing for no more than 5 sec. Centrifuge
briefly.
3.
Prepare a fresh master mix on ice according to Table 1. Mix
thoroughly and carefully by vortexing for no more than 5 sec.
Centrifuge briefly and then store on ice. Add the RNA
template in the step 5.
Note: 10 μl reverse-transcription reaction volume could be
set up for downstream qPCR. All the components can be
reduced to 1/2 to adapt for SuperReal PreMix (SYBR Green)
(FP204-01, 50 μl × 50 rxns; FP204-02, 50 μl × 200 rxns).
4.
If
setting
up
multiple
reverse-transcription
reactions,
distribute the appropriate volume of master mix into
individual reaction tubes. Keep tubes on ice.
5.
Add the template RNA (50 ng-2 μg) to the individual tubes
containing the master mix. Mix thoroughly and carefully by
vortexing for no more than 5 sec. Centrifuge briefly.
6.
Incubate for 60 min at 37°C.
7.
Add an aliquot of the finished reverse-transcription reaction
to the PCR mix.
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Note: Put the reverse-transcription products on ice first and
then apply them in following PCR amplification. For longterm storage, store the products at –20°C.
The first strand cDNA synthesis reaction mixture should not
comprise more than 1/10 of the total PCR reaction volume.
Table 1. Reverse-Transcription Reaction Components
Component
Volume/Reaction
Final Concentration
10x RT Buffer
2 μl
1×
Super pure dNTP
(2.5 mM each)
4 μl
0.5 mM each dNTP
Oligo-dT (10 μM)*
2 μl
RNA Inhibitor
1 μl
Quant Reverse
Transcriptase
1 μl
1 μM
10 U (for 20 μl
reaction system)
(for 20 μl reaction
system)
RNase-free ddH2O
X μl
Template RNA added in
the step 5
X μl
Total reaction volume
20 μl
PCR Amplification
Two Step RT-PCR
In Two-Step RT-PCR, an RNA strand is first reverse transcribed into
its DNA complement; the cDNA products can then be used for PCR
amplification. RT and PCR are preformed separately in different
tubes subsequently. Both random primers and gene specific
primers can be used in Two-Step RT PCR, according the following
steps:
1.
Synthetize the first-strand cDNA with RNA as the starting
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template according to the above procedure.
2.
Add the cDNA products into PCR reaction system. The first
strand cDNA synthesis reaction mixture should not comprise
more than 1/10 of the total PCR reaction volume.
3.
Perform the PCR amplification.
One Step RT-PCR
One step RT-PCR performs RT as well as PCR in a single buffer
system. The reaction proceeds without the addition of reagents
between the RT and PCR steps. First, cDNA first-strand is amplified
at 37°C, at which temperature the activity of Taq DNA polymerase
is pretty low. When the reverse transcription is completed,
increasing temperature inactivates the reverse transcriptase, and
Taq DNA polymerase start to synthetize cDNA.
Since the RT and PCR are performed in the same tube, the buffer
system cannot be optimized separately for these two reactions.
Furthermore, reverse transcriptase has low activity in some PCR
buffer. Therefore, One Step RT-PCR is not recommended.
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Ordering Information
RNA Isolation
Product
Size
Cat.no.
RNAprep pure Kit (For Cell/Bacteria)
50 preps
DP430
RNAprep pure Kit (For Tissue)
50 preps
DP431
RNAprep pure Kit (For Plant)
50 preps
DP432
Real–Time PCR
Product
Size
Cat.no.
SuperReal PreMix (SYBR Green)
50 μl × 50 rxns
FP204-01
50 μl × 200 rxns FP204-02
PCR
Product
Size
2× Taq PCR MasterMix (with loading dye) 1 ml
5 × 1 ml
Quant Reverse Transcriptase Handbook |
Cat.no.
KT201-01
KT201-02
6