4.7.5. Multiplex RT-(q)PCR
Often there is a need to amplify several target RNAs from a single sample. This can be done in several parallel ‘uniplex’ reactions, or in a single ‘multiplex’ reaction containing the primer pairs for all different targets (Williams et al., 1999; Wetzl et al., 2002; Syrmis et al., 2004; Szemes et al., 2002). Detection of the different amplicons is usually by size difference and electrophoresis for qualitative PCR, or by target-specific labelled probes in real-time quantitative PCR (Mackay et al., 2003). A number of such qualitative multiplex PCR protocols have been designed for honey bee viruses as well (Chen et al., 2004b; Topley et al., 2005; Grabensteiner et al., 2007; Weinstein-Texiera et al., 2008; Meeus et al., 2010). Real-time qPCR can also be multiplexed, by using a range of different fluorophores and excitation-reading laser channels. This is useful for minimizing between-reaction variability, if both target and internal reference standards can be amplified simultaneously, in the same reaction. Other uses are to distinguish between variants of the same gene or pathogen. Currently, up to four different targets can be detected and quantified simultaneously in qPCR.
However, there are many serious disadvantages of multiplexed PCR methods that may ultimately outweigh the advantages of consolidation and efficiency:
- Multiplex RT-PCR is considerably less sensitive than uniplex RT-PCR (the reagents will be exhausted by several targets instead of just one), as much as several orders of magnitude depending on the number of targets (Herrmann et al., 2004).
- Optimization of multiplex (q)PCR assays is considerably more complicated than uniplex (q)PCR, due to the large number of primers and probes that need to be optimized simultaneously for absence of undesired interactions. An alternative to multiplex RT-(q)PCR that avoids many of the assay optimization problems due to the complex primer mixes is the Multiplex Ligation Probe Amplification method, described in section 10.3.
- The PCR products need to be resolved on size or by fluorophore, before they can be quantified, nullifying many of the gains in efficiency and cost.
- Amplification (and thus quantification) of one target can be strongly affected by the prior amplification of more abundant targets in the reaction, either through competition for a limited pool of reagents, or through inhibition of the PCR reaction at later stages by the PCR products produced during earlier cycles, which sequester most of the polymerase (Santa Lucia, 2007).
- For these reasons, it is often much more practical and simple to use uniplex RT-PCR, even for large volume and throughput projects.