Master Mixes for Multiplex Real-time PCR
by Caitlin Smith
The ability to run multiplex real-time PCR (also known as or quantitative PCR or qPCR) reactions is a challenging but important endeavor. Not only does it mean higher-throughput, but it also allows researchers to use more than one reference gene for better standardizations. [1] The trick is to make the conditions just right for more than one target at a time. This is no easy feat, but can be facilitated by using qPCR master mixes, which typically consist of a pre-optimized mixture of buffer, DNA polymerase, dNTPs, MgCl2, and perhaps other proprietary additives. Also included may be a passive reference dye or fluorescent probes.
“In general, a perfect balance of qPCR efficiency, specificity, sensitivity, speed, and tolerance to a broad range of cycling conditions and sample types is yet to be achieved,” says Viresh Patel, marketing manager in the gene expression division at Bio-Rad Laboratories. “A majority of qPCR mixes are able to excel at a few of these performance criteria, but rarely at all. Of particular challenge is the ability to accommodate a variety of sample types (such as unprocessed material, limited RNA/DNA sources).” Though there is room for growth, the master mix field for multiplex qPCR is taking off. Here are some points to consider in your search for the right one for you.
Should I use probes or dyes?
Two types of molecular tools are used to detect product in qPCR reactions: probes, or intercalating dyes such as SYBR Green. Some people think probes are more specific; SYBR Green is cheaper and ideal for screening many targets. How do you decide what’s best for you if you are interested in multiplexing? “SYBR Green detection is a very cost-effective method that is mainly used to screen unknown targets,” says Annette Tietze, senior global product manager for qPCR at Qiagen. “However, when scientists find an interesting target, they tend shift to probe-based PCR because they believe probe-based assays are more specific. At Qiagen, we have compared SYBR Green and probe-based detection and have observed that with both detection methods we can achieve at least the same specificity, so there is no need to change. Nonetheless, with the increasing need for more accurate gene expression and pathogen detection, it is becoming more important to detect multiple reference genes and the target of interest in the same tube. This is only possible when probe-based multiplexing is performed.”
Aaron Nagel, senior technical services and applications consultant at Roche Applied Science, agrees that the specificity between the two methods is not markedly different, though that is the perception. “Both technologies have their own qualities,” says Nagel. “Customers sometimes think probe-based qPCR is more specific than a SYBR Green-based assay. For multiplexing, the probe-based option is the way to go, because you are looking at multiple wavelengths [from different fluorescent probes]. Technically speaking, SYBR Green is really just geared more toward a monoplex reaction.” However, one benefit of using SYBR Green is the ability to monitor the health of the qPCR reaction. Nagel believes you can take advantage of this without sacrificing probe-enabled multiplexing. “The nice thing about the SYBR Green reaction is that you are looking at all of the amplification occurring during the reaction,” he says. “You get a sense of a discreteness associated with your reaction. And whether your amplicon is discrete, or has some carry-over contamination, or has a co-amplification event associated with it. So the SYBR Green assay is a good way to evaluate primer integrity, because you are really looking at where all your reaction components are going during the course of the reaction. Of course, you really want them to go towards your target. So what we usually see, around the plateau, is primer-dimer formation, which can’t be delineated in your amplification because everything’s being amplified by the same dye. And so the SYBR Green reaction gives you an indication of the overall health of your reaction. That’s something the probed-based assay does not give you.” Nagel points out that beyond this utility, SYBR Green is not as versatile as probe-based assays, in which you can use different dyes with different wavelengths for multiplexing.
Agilent is working toward a combination approach with these technologies. “Many customers tell us that specificity, sensitivity, and speed are increasingly important as they analyze targets with fewer copy numbers, in more complex samples, and in high-throughput settings,” says Carolyn Reifsnyder, marketing director for qPCR, PCR & Bioreagents at Agilent Genomics Division. “Though Agilent offers master mixes for both SYBR Green and probes, we are working together with Integrated DNA Technologies (IDT) to deliver an integrated reagent and assay solution validated to generate accurate and reproducible results across a wide range of targets and applications. We believe the combination of Brilliant III Ultra-Faster Master Mix and PrimeTime® qPCR assays provide better efficiency and greater flexibility, which is important to customers looking for reproducible, accurate quantification at low target concentrations.” Combining a new, highly processive Taq mutant with a faster extension rate and hot-start technology, Agilent’s new Brilliant III Ultra-Fast reagents are designed for speed and sensitivity.
How much multiplexing can I gain?
Increasing the multiplexing abilities in this field would be welcome. Many vendors offer duplex reagents, and some offer reagents that accommodate up to five-plex. Agilent offers at least a 25-plex assay with their unique MassCode PCR, which combines PCR, proprietary primers, and mass spectrometric detection for nucleic acid targets. If you are not equipped for mass spectrometry, however, there are some other solutions available to give you greater than duplex capabilities.
For example, Roche Applied Science’s Light Cycler 480 Probes Master Mix can successfully support a 4-plex reaction, and has even reached up to a 5-plex reaction. “But that’s pretty rare, meaning that there are just not a lot of customers doing 5-plex-based reactions,” says Nagel. “And if they are, it’s probably not so much quantitative, as more qualitative – as in, is my target being detected, yes or no?”
Bio-Rad's iQ Multiplex Powermix allows detection of up to 4 targets when one differs in expression up to 106-fold relative to the others, and up to 5 targets under conditions of more equivalent expression.
Qiagen also offers multiplex kits for detection of up to four targets, with their new QuantiFast Multiplex PCR and RT-PCR Kits, along with their existing QuantiTect Multiplex Kits for standard cyclers. “They can be used on both standard and fast cycling instruments without the need of any extra hardware upgrades and are compatible with dual-labeled TaqMan® probes, such as QuantiFast Probe Assays,” says Tietze. “In addition, we offer Rotor-Gene Multiplex PCR and RT-PCR Kits that are dedicated multiplexing solutions for our cycler, the Rotor-Gene Q. All of these kits — the QuantiTect, QuantiFast, and Rotor-Gene Kits — are designed for gene expression analysis and the detection of up to four targets simultaneously, and can be used for both two-step and one-step RT-qPCR.”
Tietze says their kits are also especially suited for low-abundance transcripts, detecting as few as 10 copies. They are also designed to co-amplify target and endogenous controls to similar degrees, which is especially important for their pathogen detection kits, the QuantiTect Virus Kit and QuantiFast Pathogen +IC Kits. “The latter kit includes a universal internal positive control to ensure the correct interpretation of false-negative results,” says Tietze.
What can I do to increase my chances of success with multiplexing?
Start simple. Keep several things in mind to increase your chance of success when beginning multiplexing qPCR. One is to characterize your targets in a monoplex reaction before combining into a multiplex reaction. “Then, when you go into the multiplex format, you can see whether your primer-probe combinations are actually working in a monoplex fashion,” says Nagel. “You don’t want to dive in and do a 3-plex or 4-plex if your primer-probe combinations are not really good to start out with. Most vendors will offer software that allows you to select a multiplex format. This allows for compatibility, so that the software will automatically design primer-probe combinations that are compatible.”
Reaction volume.Another consideration is reaction volume. Researchers with limited and/or expensive starting materials may want to miniaturize and scale down to conserve sample. How small a reaction volume can you use without interfering in the reaction itself? “Most customers are using a 20ul reaction volume,” says Nagel. “How far can we go down? Does 5ul of reaction volume get the same results? Four times less the volume means four times less reagent. For a duplex, it is challenging because there is competition for those reagents, so you will get faster exhaustion of reagents. So you probably won’t see a lot of miniaturized, multiplex reactions because it just won’t work as well as, say, a 20ul reaction volume.” Another potential pitfall with smaller volumes is evaporation during thermocycling.
Starting material. Another multiplex consideration is the relative amounts of starting samples, as results are typically better with similar starting quantities. Bio Rad’s new patented Sso7d fusion protein technology is included in their line of qPCR supermixes, and can contribute to smoothing out this problem. “Sso7d is a small double-stranded DNA binding protein which enhances the performance of PCR polymerases by increasing enzyme speed, sensitivity, and tolerance to inhibitors,” says Patel. “These enhanced performance properties also benefit challenging qPCR applications such as multiplexing, where several targets (often varying significantly in starting amounts) must be amplified with high efficiency.” Their SsoFast Probes Supermix supports duplex reactions with starting quantities that can vary by 10,000-fold or more. It can also support a 3-plex reaction with less varied starting quantities.
Qiagen also offers a solution for varying starting quantities. “Reliable detection of both high- and low-abundance targets in the same tube is usually very difficult to achieve,” agrees Tietze. “We meet these challenges by using synthetic Factor MP, which supports macromolecular crowding and enables the amplification of both low- and high-abundant targets with the same efficiency.” She also emphasizes the importance of increasing our multiplexing capabilities in order to use proper experimental controls. “Using more than one housekeeping gene for gene expression analysis is an important requirement of the MIQE guidelines from Bustin et.al. [1],” she says. “Scientists now have the possibility to detect up to three reference genes plus a target of interest in the same tube, making it possible for them to achieve more reliable and valid results.”
Reference
[1] Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT. "The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments." Clin Chem. 55(4): 611-622, 2009.
The image at the top of this page is from Agilent's Brilliant III Ultra-Fast qPCR and qRT-PCR Master Mixes.