4plex, real-time one-step RT-PCR without optimization

Multiplex, real-time PCR allows the simultaneous quantification of multiple targets in the same reaction, and is becoming increasingly common with the introduction of cyclers that can detect different fluorophores. However, use of PCR master mixes intended for singleplex analysis often requires time-consuming optimization of primer-probe sets before satisfactory results can be achieved. In contrast, QuantiTect Multiplex Kits are specially designed for multiplex analysis, requiring no optimization of reaction and cycling conditions. We describe here the successful application of multiplex analysis to our vaccine research.

In our studies of new vaccine vectors, we used real-time RT-PCR assays to monitor the outcome of animal vaccination. This included analysis of the distribution and persistence of vaccine vectors in small animal models. In our development of a vesicular stomatitis virus (VSV) vector, we performed assays to detect vector nucleic acid and a vector-encoded gene (i.e., 2 viral RNA targets), an animal housekeeping gene (to normalize viral RNA levels), and exogenous RNA (to monitor the efficiency of RNA purification). We investigated the feasibility of analyzing these 4 RNA targets by multiplex, real-time RT-PCR. As the targets were not analyzed in separate wells, potential problems with well-to¬well variation in starting template amount and reaction conditions were avoided.

First of all, we performed an experiment using RNA standards (synthetic oligonucleotides) with the QuantiTect Multiplex RT-PCR Kit. The 4 targets were the viral nucleocapsid gene (N); the HIV-1 gene, gag (G); the mouse housekeeping gene, RPLO (R) and a spiked exogenous control, Armored-RNA (A). Multiplex, real-time one-step RT-PCR was carried out using 10-fold serial dilutions (from 10⁷ to 10¹ copies) of the viral and mouse RNA targets (N, G and R) and 10⁵ copies of the exogenous RNA (A). Reactions were run on the Applied Biosystems 7500 according to the instructions in the QuantiTect Multiplex RT-PCR Handbook. The probes were labeled with the dyes 6-FAM, VIC¨, NED and Cy¨5 for the targets N, G, R and A, respectively. The concentration of each primer or probe was 0.2 µM.

For comparison, the targets were also analyzed by singleplex, real-time one-step RT-PCR: for these reactions, primer concentration was increased to 0.4 µM, while probe concentration was kept at 0.2 µM.

The amplification plots for the 4plex assays overlapped with those for the singleplex assays (Figure 1), showing equivalent CT values at each dilution of the targets N, G, R, and A. The sensitivity of the 4plex and singleplex assays was also comparable, with detection of as little as 10 copies of the targets N, G, and R. These results demonstrate the reliability of the QuantiTect Multiplex RT-PCR Kit in multiplex, real-time RT-PCR.

Further experiments were carried out to analyze viral/mouse RNA purified from nasal turbinate homogenates by real-time one-step or two-step RT-PCR. Equivalent CT values in 4plex and singleplex assays were also observed (see reference below).

Figure 1. Comparable CT values in 4plex and singleplex assays. The QuantiTect Multiplex RT-PCR Kit was used with the Applied Biosystems 7500 to carry out 4plex, real-time one-step RT-PCR and, for comparison, singleplex, real-time one-step RT-PCR. The synthetic RNA templates were 10-fold serial dilutions (from 10⁷ to 10¹ copies) of targets N, G and R, and 10⁵ copies of target A. The amplification plots for the 4plex and singleplex assays overlapped, demonstrating the reliability of the QuantiTect Multiplex RT-PCR Kit in multiplex analysis.

QuantiTect Multiplex Kits provided specific and sensitive amplification without the need for PCR optimization, even in multiplex assays where both high-abundance and low-abundance genes were analyzed. The kits not only helped us to increase the throughput of our real-time RT-PCR assays in our vaccine research with animal models, but also reduced concerns about false-negatives. In addition, we no longer have to perform time-consuming optimization of primer–probe sets, such as limiting primer concentrations.

Visit www.qiagen.com/multiplex to find out more about QuantiTect Multiplex Kits.
Data excerpted from Coleman, J.W. et al. (2007) Simultaneous Quantification of Four RNA Targets by Multiplex, Real-Time RT-PCR without Optimization. BioTechniques 43, 369.