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Fast qRT-PCR with LightCycler® 480 RNA Master Hydrolysis Probes
Rita Haerteis, Ursula Grepl, and Waltraud Ankenbauer*
Roche Applied Science, Penzberg, Germany
*Corresponding author
Introduction
Scientists who want to increase throughput in their workflows are interested in shortening the reaction times. Fast PCR can be achieved using a fast PCR instrument like the LightCycler® 480, and the appropriate reagent like the LightCycler® 480 RNA Master Hydrolysis Probe. The enzyme component allows fast reverse transcription and the included hot-start system does not require an extra heat treatment for chemical activation. Therefore, the first two reaction steps can be shortened compared with standard protocols. In order to explore the fastest conditions possible we analyzed several variables.
The LightCycler® 480 RNA Master Hydrolysis Probes is an easy-to-use hot-start reaction mix, specifically adapted for one-step RT-PCR under the rapid and accurate cycling conditions of the plate-based LightCycler® 480 Instruments using hydrolysis probes (e.g., Universal ProbeLibrary probes) as detection format. The kit provides reagents, including an RNA master mix (with buffer, nucleotides, and enzyme), a Mn(OAc)2 stock solution, PCR-grade water, and enhancer solution. The LightCycler® 480 RNA Master Hydrolysis Probes can be used in conjunction with heat-labile Uracil DNA Glycosylase for carryover prevention during PCR.
Materials and Methods
The experiments were performed with HeLa total RNA in dilutions from 10 ng to 0.1 pg (A, standard protocol), and 100 pg to 0.1 pg for protocols B to D. Primers directed to ABCC2 RNA were used and the products were detected with a Universal ProbeLibrary probe.
Results and Discussion
In protocol A we used a fast RT step and a conventional PCR protocol. In protocols B and C the RT step times were reduced. In protocol D the incubation times for the PCR cycles were drastically reduced. In order to check whether contaminating DNA was detected rather than a product derived from the RNA, a control reaction was performed with the same RNA, primer and probe but using LightCycler® 480 Fast DNA Master. This control reaction did not result in any significant amplification (not shown).
As shown in Table 1, the cp values depicted for the 100-pg and 10-pg template concentrations are quite similar. LightCycler® 480 RNA Master Hydrolysis Probes amplified the RNA to the same extent and similar sensitivity independent of the amplification protocol chosen (Figure 1 and Table 1). Thus, a very short total run time can be achieved and more than 55% time saved (Figure 2).
A comparison of the reaction times of protocol D with those recommended for a competitor product is depicted in Figure 3. The competitor system requires nearly twice the reaction time the LightCycler® 480 System requires.
This article was originally published in Biochemica 2/2008, pages 21-22. ©Springer Medizin Verlag 2008
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