Editorial Article
Wednesday August 18, 2010
by Caitlin Smith
Multiplexing your real-time or quantitative PCR reactions would save time and work – but are you up for the challenge? The resources are out there to help you tackle this adventure, and you won’t be alone. “The general increase in the number of users who are attempting multiplexing is exciting,” says David Moore, senior product manager for qPCR instrumentation at Stratagene, a division of Agilent Technologies. Even though multiplexing can be rough-going at first, sticking with it can earn you tremendous experimental rewards. “It can be difficult to optimize an assay for multiplexing, but with the right instrument and software, it can be a powerful technique.”
Flexibility with filters
Your multiplexing abilities will be greatly enhanced by filtering the excitation light properly. Agilent’s Mx3005P qPCR instrument is designed for flexibility in filter choices for multiplexing. . “Researchers can choose from a list of eight filter sets that span the spectrum thus allowing the use of deep blue to far red dyes," says Moore. "The excitation and emission filters are defined to narrow wavelengths to minimize fluorescent crosstalk ,which allows researchers to select 5 distinct filter sets with minimal overlap to conduct effective multiplexing.”
Another system providing flexibility is Life Technologies’ new ViiA™ 7 Real-Time PCR System, which allows users to choose up to 21 unique filter combinations for multiplexing. “We have reagent products that support multiplexing for bacterial detection, virus detection, and gene expression,” says Rob Tapella, associate product manager for real-time PCR reagents at Life Technologies. “For example, with the new TaqMan® Fast Virus 1-Step Master Mix, we have created a simpler one-step qRT-PCR reagent by merging all of the components into a single tube, including the reverse transcriptase. This product is also very robust to PCR inhibitors commonly found in samples used by virus researchers. Often, these researchers prefer to multiplex targets so that several viruses can be screened for simultaneously in a sample, so we have designed the mix to support that as well.” In addition to the TaqMan® Fast Virus 1-Step Master Mix, Life Technologies just released their TaqMan® Fast Advanced Master Mix, which shortens run times for qPCR reactions.
Bio-Rad’s CFX96 and CFX384 real-time PCR systems also filter the excitation light and resulting signal, which eliminates “cross-talk” between dyes in multiplex reactions, according to Rachel Scott, senior product manager in the gene expression division at Bio-Rad. “The 6-channel scanning optics shuttle of the CFX passes directly over every well ensuring that every sample across the block receives uniform excitation conditions and data collection is consistent for every well,” says Scott, noting that new developments in epigenetics and sequencing are changing our understanding of gene regulation. “Multiplex PCR offers a powerful complementary technique to explore gene organization, insertions, deletions, polymorphisms, repeats and their relationship to disease with increased confidence through the inclusion of internal controls.”
Designing against problems
One of the more immediate challenges facing multiplexers is non-specific interactions, and designing primer-probe sets. “Especially once we reach three, four, or more targets that are being assayed simultaneously, it can be a challenge to prevent non-specific interactions between the components,” says Tapella. “This is mainly a primer design issue which takes either specialized software, or diligence and experience to be sure that primer-primer interactions and folding will minimize impact on the performance of the assays.”
An example of specialized software is Biosearch Technologies’ online tool. “The Biosearch Technologies website features RealTimeDesign™ software, which includes a module for multiplex assay design,” says Ryan Brady, marketing specialist at Biosearch Technologies. “Even with such powerful software tools easing the design process, assay validation remains a challenge. When performing a singleplex assay, targets often have quirks that are resolved without too much trouble. In a multiplex assay, the small challenges associated with each target have a combinatorial effect which can amount to a significant problem for validating a multiplexed experiment.”
In addition to primer-primer interactions, Roche Applied Science’s marketing manager Larson Manifold notes the importance of primer-template interactions. “You want the primers to lay down in the right place on the right gene, so you have to make sure that they’re not homologous to other areas, or other sequences, on your transcript,” he says. “And then you have probe-template interactions – so you have to ensure that your probe is specific to a sequence within the target gene, and only one target gene, and not more than one target gene.” Manifold continues that “when you consider probes, primers, and templates, and start adding up the number of possibilities of interactions between these three different elements, in a singleplex reaction you have x number of interactions. In a duplex reaction, you have another order of magnitude higher. The more targets you’re going after in the same reaction, the more complicated the reaction set-up becomes, the greater chance of unintentional amplification and detection you get.” Roche Applied Science’s Universal Probe Library, a set of hydrolysis probes used for mRNA assays, has recently grown with the inclusion of reference gene assays for running a reference gene and a target gene expression assay in the same well. In addition, Roche’s LightCycler 480 instrument can run up to 6-plex qPCR reactions.
Scott agrees that the biggest challenge is ensuring that multiplex results are “accurate reflections of gene expression, and not affected by competition for reaction components or poor fluorophore de-convolution,” she says. “Multiplex reactions should ideally be validated against the results from a robust and efficient simplex reaction to ensure reliable and repeatable data is generated.”
Reaction reagents can be especially useful in preventing problems. “The next challenge is specific to situations in which all targets are measured quantitatively and all targets are present -- if the reaction conditions are not appropriate, higher concentration targets can cause problems quantifying the lower concentration targets, and can also decrease sensitivity,” says Tapella. “This is where using an appropriate master mix or other reagent is important.”
Dyes to meet the challenge
Even though the past decade has seen some remarkable engineering of fluorescent dyes in general, multiplexers are still clamoring for more. With more channels in demand, they now want fluorescent probes with narrower emission spectra so that they won’t overlap with their reaction-mates, thereby allowing researchers to measure separate signals for each one – not to mention avoiding undesirable artifacts. “The final challenge has to do with the fluorescent dyes available to perform a multiplex real-time PCR,” says Tapella. “Many dye emission spectra will bleed into multiple read channels in real-time PCR instruments and cause spurious detection events or incorrect quantification results. As a system provider, we are working on this problem with both improved calibration methods as well as next-generation dyes that give better spectral separation on our instruments, such as the 7500 System and the new ViiA 7 System.”
Biosearch Technologies recently broadened their line of BHQplus™ (Black Hole Quencher®) probes to include several different fluorophores. “BHQplus probes are notable for their ability to quantify difficult targets, and this new development opens up the possibility of using BHQplus probes for a multiplexed assay,” says Brady.
It looks as if there might be light at the end of this tunnel that will help to further the multiplex adventure for many more qPCR researchers new to multiplexing. “The real developments have actually come from generating new fluorophores that don’t bleed-through into different channels, in terms of detection filters,” says Rama Badugu, technical specialist for Roche Applied Science. “We are starting to see more fluorophores with a narrow emission that can be detected in one channel.”