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cMasterTM RTplusPCR System for high flexibility in RT-PCR
 
 
Applications



Lars Peters (Eppendorf–5 Prime)
Andrés Jarrin (Eppendorf AG)


Introduction

Reverse Transcription followed by PCR1 (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 have to be analysed in parallel while the Two Step protocol is used for difficult targets or multiple target amplification from a single cDNA pool. The new Eppendorf cMasterRTplusPCR System enables the full range of RT-PCR applications. A single kit combines One- and Two Step RT-PCR protocols with high dynamic detection ranges and broad ranges of PCR product size.

The following set of experiments was designed to demonstrate the use of the kit in various applications.


Experiment 1: One Step, Sensitivity Test

Experiment 1 focuses on the incredible sensitivity of the new system when it comes to One Step RT-PCR. Working with small amounts of target material is the most critical factor in One Step applications.

Target for RT-PCR amplification: human Alpha Tubulin mRNA, a 500 bp fragment. Template RNA: total RNA purified from human HELA cells. Template concentrations in the reactions ranged from 10 pg down to 1 µg.

Protocol: Eppendorf cMaster RTplusPCR System, standard One Step RT-PCR protocol. 50 µl reations in 1:10 diluted RTplusPCR Buffer at 2.5 mM final magnesium concentration.


Materials

- Eppendorf Perfect RNA Eukaryotic Kit
- Eppendorf cMasterRTplusPCR System

PCR reactions were performed on the Eppendorf Mastercycler gradient.


Set-up of One Step RT-PCR reactions

Components RT-PCR Volume 50 µl Final concentration in the reaction
RNase-free H2O 15.1 µl
dNTP mix [10 mM each] 1.5 µl 300 µM
Tubulin forward primer 0.2 µl 200 nM
Tubulin reverse primer 0.2 µl 200 nM
Total HELA RNA 3 µl 1 µg –10 pg in 50 µl
Master mix 1 20 µl
RNase-free H2O 24 µl
RTplusPCR Buffer with Mg2+ 5 µl 1x; 2.5 mM Mg2+
cMaster™ RTEnzyme 0.5 µl 0.15 U/µl
cMaster™ PCR Enzyme Mix 0.5 µl 0.05 U/µl
Master mix 2 30 µl





Cycling program parameters

Cycle Step Temp. Time Description
1x 1 50ºC 30 min Reverse transcription
1x 2 94ºC 3 min Initial denaturation
40x 3 94ºC 15 sec Template denaturation
40x 4 68ºC 45 sec Primer annealing / elongation


Results Experiment 1: Sensitivity of One Step RT-PCR



Fig. 1: One Step RT-PCR amplifikation of an Alpha Tubulin cDNA fragment from various amounts of total RNA.

Lane 1: 1 µg
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 down to 10 pg of total RNA.


Discussion
The cMaster RT is designed to provide maximum flexibility for all demands of RT and RT-PCR. A recombinant homodimeric viral reverse transcriptase together with a unique formulation of the RTplusPCR buffer allows efficient cDNA synthesis over a broad temperature range (37– 60 °C) and product size (0,2–12,5 kb). The cMasterRTplusPCR 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 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 (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. hybridisation experiments.