RT-PCR has become an essential method that allows researchers to determine
the presence, structure and level of expression of an mRNA transcript in
the organism, tissue or cell of interest. The basic protocol involves the
synthesis of a cDNA copy of all the mRNA present in the sample, followed
by amplification of the gene(s) of interest. Due to buffer incompatibilities,
the two steps (RT and PCR) are often performed separately. While there are
kits that combine the two steps into one, the limitations on sensitivity,
due to the aforementioned buffer incompatibilities, are great. Eppendorf
has designed a new kit with novel chemistries that allow for a more robust
single-step RT-PCR reaction and even greater sensitivity in Two Step experiments.
This paper examines the performance of this new kit.
Reverse Transcription followed by PCR* (RT-PCR) is one of the most popular
techniques for RNA analysis. One Step RT-PCR is the method of choice if
many RNA samples need to be analyzed in parallel; the Two Step protocol
is used for difficult targets or multiple target amplification from a single
cDNA pool. The new Eppendorf cMaster RTplusPCR System enables the full range
of RT-PCR applications. It contains a novel, recombinant reverse transcriptase,
and a high fidelity PCR enzyme mix. In addition, the RTplusPCR buffer is
formulated to adjust the Mg2+ concentration automatically, so that there
is never a need for optimization of this critical component. This is achieved
by weakly chelating Mg 2+ ions: when Mg2+ is present in excess, it is bound
by the chelating agent, but as it is needed by the reaction (for Taq, or
DNA), it is re
leased. This novel technology increases the sensitivity and
specificity of cMaster RTplusPCR. A single kit combines One- and Two Step
RT-PCR protocols with high dynamic detection ranges and broad ranges of
PCR product size (up to 5.3 kb for One Step reactions; up to 12.3 kb for
Two Step protocols). The following experiments were designed to demonstrate
the use of the kit in various applications.
The first experiment focuses on the extraordinary sensitivity of the new
system with respect to One Step RT-PCR. Working with small amounts of target
material is the most critical factor in One Step applications. Performing
long range RT-PCR in a One Step protocol is no longer a problem as demonstrated
in experiment two. The novel buffer system in the kit allows optimal processivity
of both the reverse transcriptase and proofreading polymerase mix. The optional
Two Step RT-PCR protocol provided in the system is adaptable to a wide range
of templates. Experiment three depicts amplification of various templates.
Whether moderately sized or very long, low- or high abundance, the new system
offers a comprehensive
solution for all RT targets.
Materials and Methods
Eppendorf cMaster RTplusPCR System
Eppendorf Perfect RNA Eukaryotic Kit
All PCR* reactions were performed on the Eppendorf Mastercycler gradient.
Experiment 1: One Step, Sensitivity Test
Target for RT-PCR amplification: human Alpha Tubulin mRNA, a 500 fragment.
Template RNA: total RNA purified from human HELA cells. Template concentrations
in the reactions ranged from 1 g down to 10 pg. Protocol: Eppendorf
cMaster RTplusPCR System, standard One Step RT-PCR protocol. 50 l
ions in 1:10 diluted RTplusPCR Buffer at 2.5 mM final Magnesium concentration.
Set-up for One Step RT-PCR reactions
Cycling program parameters
Experiment 2: One Step, Long Range
Target for RT-PCR amplification: human Tuberous Sclerosis Factor (hTSF)
mRNA, a 5.3 kb fragment. Template RNA: total RNA purified from human HELA
cells, template concentrations in the reactions are 100 and 10 ng.
Protocol: Eppendorf cMaster RTplusPCR Kit, special One Step
RT-PCR protocol for long templates.
Set-up for One Step RT-PCR reactions
Cycling program parameters
Experiment 3: Two Step RT-PCR
Targets for Two Step RT-PCR amplification:
Alpha Tubulin mRNA (mouse and human), 500 bp fragment
Tumor Necrosis Factor Receptor (TNFR1) mRNA (mouse and human), 1.3 kb fragment
Human Tuberous Sclerosis Factor (hTSF) mRNA, 5.3 kb fragment
Mouse Dynein mRNA, 12.3 kb fragment
Protocol: Eppendorf cMaster RTplusPCR Kit, Two Step RT-PCR
protocol for standard sized and long templates (manual). All reverse
transcription reactions were primed with 0.5 g Oligo(dT)20 primer.
Set-up for the first step RT reaction
Set-up for the second step PCR reaction
Set-up for RT reaction
Two step cycling program used for Tubulin and TNFR1
Three step cycling program used for hTSF and Dynein
Target size, elongation time and annealing temperatures used
Tubulin forward: CACCCGTCTTCAGGGCTTCTTGGTTT
Tubulin reverse: CATTTCACCATCTGGTTGGCTGGCTC
TNFR forward: CCACCATCTCGGTCATCAGGATTGCCT
TNFR reverse: TTCTCATGGAAGCTATGGGTATCACAT
hTSF forward: GGAGTTTATCATCACCGCGGAAATACTGAGAG
hTSF reverse: TATTTCACTGACAGGCAATACCGTCCAAGG
Dynein forward: CGGCGCTGGAGGAGAA
Dynein reverse: AGGTGGTGGCTCAAACACAAAG
Experiment 1: Sensitivity of One Step RT-PCR
Fig. 1: One Step RT-PCR amplification of an
Alpha Tubulin cDNA fragment from various
amounts of total RNA.
Lane 1: 1 g
Lane 2: 100 ng
Lane 3: 10 ng
Lane 4: 1 ng
Lane 5: 100 pg
Lane 6: 10 pg
M: 100 bp ladder (NEB)
The cMaster RTplusPCR System shows a high dynamic range of product yield
depending on the amount of template RNA. Alpha Tubulin mRNA can be detected
in HELA RNA down to 10 pg of total RNA.
Experiment 2: Long range One Step RT-PCR with various amounts of total
Fig. 2: Long range One Step RT-PCR of the human
Tuberous Sclerosis Factor mRNA.
M: DNA Ruler LadderMix 0.110 kb (MBI Fermentas).
Lane 1: 100 ng total RNA
Lane 2: 10 ng total RNA
Lane 3: 0 ng total RNA (negative control)
As shown in Fig. 2, a 5.3 kb hTSF PCR product is de
tectable in 100 and 10
ng of total RNA using the One Step RT-PCR protocol. This is a very difficult
PCR at best, and most manufacturers of RT-PCR kits do not even publish results
of one-step protocols on templates this long. In contrast the Eppendorf
kit can consistently and reliably amplify this product from 10 ng of RNA
from HELA cells.
Experiment 3: Two Step RT-PCR of short, long, rare and abundant transcripts
from various sources of total RNA.
Fig. 3: Two Step RT-PCR of multiple targets and RNA sources. M: DNA Ladder
Mix 0.110 kb (MBI Fermentas).
RT-PCR was performed on five genes that exhibit various levels of expression.
In order to maximize sensitivity the RT reaction was performed separately
from the PCR reaction. In the case of Alpha Tubulin and TNFR1, we were able
to use a two-step protocol and combined the annealing and elongation step
into a single 68 C step. As shown in fig. 3, all reactions worked well
regardless of fragment length or copy number. Even Dynein, an extremely
rare transcript that is
very long (12.3 kb), was efficiently amplified with the cMaster RTplusPCR.
These results were extremely reproducible and show that the Eppendorf kit
can be expected to give reliable results for difficult RT-PCR reactions.
Details for Fig. 3
The cMaster RT is designed to provide maximum flexibility for all demanding
RT and RT-PCR applications. A recombinant homo-dimeric viral reverse transcriptase
together with a unique formulation of the RTplusPCR buffer allows efficient
cDNA synthesis over a broad temperature range (37 C60 C)
and product size (0.2 kb12.5 kb). The cMaster RTplusPCR enzyme mix
uses a blend of thermostable DNA polymerases with proofreading assisted
fidelity and high extension rate. Performing the One Step procedure, sensitive
detection out of smallest amounts of total RNA and amplification of up to
5.3 kb cDNAs is
possible (see Fig. 1 and Fig. 2). The Two Step protocol enables multiple
cDNA targets to be amplified from an RT reaction, extending the PCR product
size up to 12.3 kb (see Fig. 3). To further extend flexibility, the RT-only
option of the system can be used to combine cMaster RT with any PCR system
or to generate cDNA probes for other downstream applications, e.g. hybridization
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