Multiplex, real-time PCR — a powerful technique in drug discovery

By Mark Richards, Senior Marketing Manager – Pharma, Europe and North America

Multiplex, real-time PCR extends the power of PCR technology to provide quantitative data for multiple sequences of interest in a single-tube reaction. We examine the advantages of this technology, and describe its application in drug discovery.

Real-time PCR involves measurement of amplified DNA using fluorescence as the reaction progresses in real-time. Multiplex, real-time PCR is an extension of the real-time PCR technique in which multiple targets are quantified simultaneously in the same reaction. It is based on the use of probes labeled with distinct fluorescence dyes and real-time cyclers that allow the excitation and detection of several non-overlapping dyes in the same well or tube.
Real-time PCR is widely used in drug research and development for applications including genotyping, vaccine studies, and the discovery and validation of biomarkers.     In recent years, drug discovery and development has begun to target specific patient populations, with the goal of increasing efficacy of new therapies while limiting potential adverse effects. The achievement of such personalized healthcare is dependent on the identification of biomarkers: biological features that can be used to identify disease type or measure disease progress. Biomarkers are also used to increase the success rate and assist regulatory approval of clinical trials by targeting population groups likely to respond to the therapy under study and excluding potentially nonresponsive populations.

Here we describe how multiplex, real-time PCR can be used to facilitate biomarker and vaccine studies by increasing reliability and reproducibility of results, which is critical for drug discovery studies.

Benefits of multiplex, real-time PCR

One of the major benefits of multiplex, real-time PCR is the ease and reliability of normalization. Normalization enables relative quantification, which is a commonly used method of quantification in real-time experiments. The target gene(s) and reference gene(s) (e.g., a housekeeping gene) are amplified, and the expression of the target gene is normalized to that of the reference gene. Normalization provides an internal control that corrects for factors that could otherwise lead to inaccurate quantification including variation in quantity of input sample, sample degradation, presence of inhibitors, and differences in sample handling. Normalization also allows results from different experiments and samples to be compared directly. Using multiplex PCR, target and reference genes can be amplified in the same reaction, eliminating the well-to-well variability that would occur if separate amplification reactions were carried out, thereby further increasing precision and reliability. Additional advantages of multiplex PCR include reduced reagent costs, conservation of precious sample material, time savings, increased throughput, and increased results per sample.

Multiplex PCR increases reproducibility of results

Data from Angelika Meyer (Novartis Pharma AG, Basel, Switzerland) demonstrates how amplification of reference and target genes in the same reaction, instead of separate reactions, increases the reliability of gene quantification. In the experiment shown in Figure 1, myogenin expression was examined in untreated cells or after treatment of cells with one of two compounds. The housekeeping gene GAPDH was used as a reference gene and was amplified in the same reaction as the myogenin gene. In 5 independent experiments, changes in myogenin expression were detected with high reproducibility.

These results show how multiplex, real-time PCR can provide the level of reproducibility that is essential for studies of drug response or drug dosage effects on gene expression. A similar experimental scenario could be applied to biomarker identification and validation experiments, in which the effect of a drug on expression of a genetic marker is studied.

Figure 1. Reliable relative quantification. Cell line X was treated with one of 2 compounds (Y or Z) or left untreated (U). RNA was purified and duplex, real-time one-step RT-PCR was carried out using the QuantiFast Multiplex RT-PCR +R Kit and TaqMan Gene Expression Assays for myogenin and GAPDH. For each treatment, 5 independent experiments were performed. Changes in myogenin expression were detected with high accuracy and reproducibility (green curves). The expression of the housekeeping gene GAPDH was similar in all experiments (blue curves), allowing normalization of myogenin expression levels using the ΔΔCT method of relative quantification. The fold changes in normalized myogenin expression level relative to that in untreated cells were consistent between experiments, as indicated by the small error bars.

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

John W. Coleman, J. Erik Johnson, and David K. Clarke (Wyeth Vaccines Research, now part of Pfizer) used real-time RT-PCR assays to monitor the outcome of animal vaccination. Their studies involve analysis of the distribution and persistence of vaccine vectors in small animal models. In their development of a vesicular stomatitis virus (VSV) vector, they 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). They investigated the feasibility of analyzing these 4 RNA targets by multiplex, real-time RT-PCR by performing experiments comparing 4plex and singleplex real-time RT-PCR of synthetic RNA oligonucleotides. The amplification plots for the 4plex assays overlapped with those for the singleplex assays and the sensitivity of the 4plex and singleplex assays was also comparable, with detection of as little as 10 copies of the targets (see Figure 2).

Figure 2. Comparable CT values in 4plex and singleplex assays. The QuantiTect Multiplex RT-PCR Kit was used 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 (the viral nucleocapsid gene), G (the HIV-1 gene, gag), and R (the mouse housekeeping gene, RPLO), and 105 copies of target A (a spiked exogenous control, Armored-RNA). The probes were labeled with the dyes 6-FAM, VIC, NED, and Cy5 for the targets N, G, R, and A, respectively. For 4plex assays, the concentration of each primer or probe was 0.2 µM. For singleplex assays, primer concentration was increased to 0.4 µM, while probe concentration was kept at 0.2 µM. The amplification plots for the 4plex and singleplex assays overlapped, demonstrating the reliability of the QuantiTect Multiplex RT-PCR Kit in multiplex analysis.

Use of multiplex real-time RT-PCR provided several critical advantages for these vaccine studies. 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. The kit used (QuantiTect Multiplex RT-PCR Kit, QIAGEN) provided specific and sensitive amplification without the need for PCR optimization even in assays where both high-abundance and low-abundance genes were analyzed. The technique increased throughput, but also reduced concerns about false-negatives. In addition, use of the kit eliminated the need to perform time-consuming optimization of primer–probe sets, such as limiting primer concentrations.

Technology of choice for drug development

The most important benefit offered by the multiplexing technique is the accuracy of results due to amplification of multiple target and reference genes in a single reaction. Elimination of sample handling errors and well-to-well variation mean that multiplex PCR is increasingly accepted as the technology of choice for gene expression analysis in a drug discovery setting. Applications of real-time, multiplex PCR include discovery of biomarkers and gene signature analysis in the context of drug development and clinical studies. In addition, this technique will contribute to personalized healthcare and enable the development of innovative and targeted new therapies. For a list of publications using multiplex, real-time PCR, visit the QIAGEN Reference Database.