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qPCR Goes Plural

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As quantitative PCR has continued to improve, so too has multiplexed qPCR — an approach that combines several PCR assays into one to increase throughput and conserve sample material. Multiplexed qPCR works by simultaneously amplifying and independently detecting targets with unique spectra. While it does require more time and effort to implement than single-plex qPCR, users are compensated with rich data and a cost-effective way to repeatedly run an optimized assay.

Many vendors, including Agilent, Bio-Rad Laboratories, Qiagen, and Roche Applied Science, offer reagents that can accommodate multiplex assays — Roche's Light Cycle 480 Probes Master Mix can support up to a five-plex reaction and Agilent offers a 25-plex assay with its MassCode PCR solution, which consists of PCR, primers, and mass spectrometry detection for nucleic acid targets.

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"There is an increasing need to look at multiple markers within the same sample, particularly in the discovery and validation of biomarkers, which will lead to new diagnostic applications," says Darren Link, vice president of research and development at RainDance Technologies. "There are also cost, throughput, sample preservation, and quality of results reasons to multiplex versus splitting the sample and running it as a number of single-plex reactions."

Perfect conditions

One aspect of multiplex qPCR that still requires some development is the reaction conditions, namely to ensure that they work for more than one target. One option for creating that ideal balance is to use qPCR master mixes that are essentially a mixture of buffer, DNA polymerase, dNTPs, and magnesium chloride, and the mixes can also include a passive reference dye or fluorescent probes. There are qPCR mixes that can satisfy the need for efficiency, sensitivity, resilience to cycling conditions and various samples, or speed, but no one qPCR mix does it all while preventing inaccurate measurements from sample to sample.

"The main challenge in optimizing multiplex qPCR is making sure the combination of probe dyes and quenchers, assay-target sequences, master-mix reagents, and instrument platform are all compatible," says Allen Nguyen, senior manager of qPCR Strategic Solutions at Life Technologies. "While this sounds straightforward, researchers have traditionally run into challenges when they have tried to multiplex multiple assay targets due to unknown factors, such as the concentration of each individual target in the sample and its disproportionate utilization of reagents, or the concentrations of the primers in the qPCR reaction not being optimized."

Go digital

While there are means for controlling the competition for reagents between the simultaneous reactions in multiplexing qPCR, it can only be managed to a certain extent. This is why some investigators say that a better option than multiplexing alone — unless required for regulatory reasons — is to run digital PCR. With digital PCR, users looking to identify rare mutations, quantify viral loads, gDNA, cDNA, plasmids, and next-generation sequencing libraries have a highly accurate and sensitive method.
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"In digital PCR, the sample is distributed into large number of reaction containers that each only contain, maximum, one molecule. This eliminates the competition for reagents, and multiplexing becomes trivial and, in fact, even higher order multiplexing is possible," says Mikael Kubista, CEO of the TATAA Biocenter. "Supported by Life Technologies, we have opened a digital PCR service in Europe based on their OpenArray platform, and multiplexing is one of the most popular requests."

The TATAA Biocenter is one of the largest certified qPCR service providers in Europe and has two locations that offer multiplex qPCR solutions. One in Gothenburg, Sweden, offers the Life Technologies OpenArray platform, which has 3,072 reaction chambers per plate with a three-plate simultaneous run capability. At the other facility, in Prague, TATAA provides digital PCR services on Fludigm's BioMark system, which has one 12.765 chip array and one 48.770 chip array.

In May, RainDance's Link and his colleagues published a paper in Lab on a Chip that detailed their efforts to expand multiplex digital qPCR by breaking the one-target per colored fluorescent probe barrier. The authors describe a novel protocol for multiplexing dPCR in picoliter droplets within emulsions that work by capitalizing on both the high number of reactions within emulsions and the high probability that only a single target DNA molecule will be activated within each droplet. By adding multiple colors, the number of possible reactions is increased geometrically, instead of linearly, as is the case with standard qPCR. In a pilot study using samples from spinal muscular atrophy patients, the team demonstrated a five-plex assay with two fluorophores could measure the copy number of genes SMN1 and SMN2 simultaneously as well as genotype a single nucleotide polymorphism in SMN1.

"Developing robust multiplex applications with qPCR has traditionally been challenging, so many researchers have avoided it," Link says. "But a multiplex digital PCR assay is much easier to prepare and more robust than a multiplex qPCR reaction because the limiting dilution of the input DNA makes each reaction a single molecule reaction that cannot interfere with a second product in the same droplet."

The major challenge in design and implementation of multiplex dPCR is to have a sufficient number of reactions. Right now, Link says that dPCR is currently of enormous interest for both academic and industrial development labs, and creative concepts are coming from multiple sources. "The simple fact that there will be choices and new solutions will help speed the adoption rate," he adds.

New ways

Some of these new solutions have already begun to appear in the literature, as academic qPCR developers look for ways to apply multiplexing to their work. One such method, for measuring telomere length, was introduced by researchers at the University of Utah in 2009. That approach continues to be implemented. Called monochrome multiplex qPCR, the method uses a single fluorescent DNA-intercalating dye that reduces measurement variability — a key issue with any type of multiplexing. Monochrome multiplex qPCR has been used to ramp up lung cancer research by aiding in studies aimed at elucidating -telomere measurements in peripheral white blood cell DNA, and could serve as an accurate predictor of lung cancer.
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A recent study led by investigators at the National Cancer Institute's Division of Cancer Epidemiology and Genetics used monochrome multiplex qPCR to compare telomere length relative to standard DNA in white blood cell DNA in 229 lung cancer cases and 229 matched controls within the prospective Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study of male smokers. The team found that longer telomeres might be associated with an increased risk of lung cancer among male smokers.

Another flavor of multiplex qPCR that has proven its value as a diagnostic tool is allele-specific methylated multiplex real-time quantitative PCR, or ASMM RTQ-PCR. One team recently demonstrated the effectiveness of ASMM RTQ-PCR in diagnosing Beckwith-Wiedemann syndrome — which is characterized by fetal overgrowth and high tumor growth risk — and Russell-Silver syndrome, characterized by intrauterine and post-natal growth restriction. Prior to the application of ASMM RTQ-PCR, clinical labs used Southern blotting to diagnose both Russell-Silver and Beckwith-Wiedemann syndromes. But the qPCR method was shown to be not only more sensitive than Southern blotting, but also a rapid, reliable, and cost-effective diagnostic method.

"Before the development of ASMM RTQ-PCR assay, we were using Southern blotting for the molecular diagnosis of RSS and BWS, however it was laborious and time consuming — sometimes taking 15 days to have the results — as well as requiring large amounts of DNA and the use of radioactivity," says Irène Netchine, a researcher and physician at the Hôpital Armand-Trousseau in Paris. "By contrast, ASMM RTQ-PCR is as powerful as Southern blotting for the study of imprinting disorders and more sensitive than Southern blotting and MS-MPLA, especially for the detection of loss of imprinting at a low degree of mosaic ... and it requires small amount of DNA. All these advantages make ASMM RTQ-PCR incontestably a better alternative than Southern blotting."

To develop a multiplex assay for studying such rare diseases, researchers face the daunting task of designing the appropriate primers and probes specific to each locus, as vendors offerings are relatively limited for this purpose. Since Netchine and her team first figured out the chemistry to design their own primers and probes, they have never looked back. "For the specific field of imprinting disorders, so far, there are many multiplex solutions available from commercial vendors," she says. "Regardless, ASMM RTQ-PCR is a suitable technique for the validation of results from whole genome methylation studies and, in our lab, we now only use ASMM RTQ-PCR to search for imprinting defects both in diagnosis and research."

The grid format of a multi-well plate can also be used to measure mRNA levels across an entire tissue sample. A group of qPCR developers from the University of Maryland are working on a 2D reverse-transcriptase-qPCR method that macro-dissects the tissue sample and preserves the actual location of the RNA, doing away with the standard homogenized approach of grinding up an entire tissue sample that only provides an average of the whole tissue. A purification protocol is then performed in the multi-well plate to prepare the samples for RT-qPCR analysis.
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"Our aim was to be able to map mRNA across tissue sections and, in particular, low-abundance mRNA that needs to be amplified to be detected, so that you can eventually correlate the mRNA position with the locations of tumors or particular types of tissues. So to maintain the two dimensional spatial information from the tissue section, to not grind up the whole tissue section and just average it, but to actually know what was going on as a function of division in a tissue," Elisabeth Smela, a professor at the University of Maryland. "It's similar to in situ PCR, except that attempting to do the amplification within the tissue, which often causes that process to fail, we take the mRNA out of the tissue we amplify in an aqueous environment."

In May, Smela and her colleagues published data in Analytical and Bioanalytical Chemistry on the effectiveness of their method for analyzing several tissue types. They are now trying to create even higher resolution maps. One goal of their project is to be able to implement their method so that the PCR process can be multiplexed.

"We were doing it in a multi-well plate so the pixels, so to speak, were too large. What we have shown is that we can do all of the reactions in a single well, and now we need to scale it down. The technique as it exists now is being applied to study more tissues," Smela says. "Because if we wanted to map what is happening in terms of gene expression across a tissue section, we would like to get down to the sub-millimeter and even to below 100 microns. The miniaturization is challenging, and we need to figure out how to do that so that we can make high resolution maps."

Words of caution

As new multiplexing qPCR platforms appear, some qPCR veterans warn that the technology needs to be approached with pragmatism. "The idea of multiplex qPCR is very enticing; however, in practice, it can be a challenge to pull off correctly. You have to be able to show that the assays running in the multiplexed reaction are behaving the same or very closely to how they perform when run in isolation," says Gregory Shipley, director of the Quantitative Genomics Core Laboratory at the University of Texas. "There are software packages out there that can help in designing multiplexed reactions, such as AlleleID from Premier Biosoft, but even then you have to do the lab work to show that everything is working."

Unless there is an assay combination that will be used many times over a long period of time, Shipley recommends that his customers stick with a single reaction per well. "If it would be worth investing the time to develop the multiplexed assay because there are cost savings and higher throughput as rewards, the newer filter-based qPCR instruments can multiplex four to six different fluorophores at one time and have software to negate spillover from one assay signal into another so the instruments are not the limiting step," Shipley says. However, he adds that "the bottom line [is that] it's good idea if you will use the multiplex a lot over time, but it can be challenging to put into practice."

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