A subsegment of the qPCR market, nanoliter qPCR technologies have expanded qPCR capabilities and applications, enabling thousands of assays to be performed simultaneously with smaller sample amounts, resulting in lower reagent costs, higher throughput and more data points. Nanoliter PCR formats were introduced in the late 2000s and initially aimed at the largest application segments of the qPCR market: gene expression and genotyping. For such applications, qPCR is considered the gold standard, providing the highest sensitivity and widest dynamic range. An example of the technology development to accommodate smaller sample volumes and a greater number of samples, nanoliter qPCR for gene expression and genotyping continue to advance.

Thermo Fisher Scientific, the world’s largest provider of qPCR systems and reagents, through its Life Technologies business, has offered its OpenArray nanoliter qPCR technology since 2009. Initially available through a collaboration agreement with BioTrove, which Life Technologies acquired (see IBO 11/15/09), OpenArray technology consists of a microscope slide–sized steel plate with 3,072 holes for individual reactions, or the equivalent of eight 384-well plates. The plates are available prefilled with either customized content or in preconfigured formats with flexible or fixed content. Thermo currently offers five formats for gene expression assays and six formats for genotyping. Samples are automatically loaded onto the plates using the QuantStudio 12K Flex AccuFill System. Plates are analyzed using the QuantStudio 12K Flex Real-Time PCR System, which can run up to four plates at a time and can be used for end-point and digital PCR.

In addition to the OpenArray format, the QuantStudio 12K system also accommodates TaqMan Array Cards, and 96- and 384-well plates. As Ramesh Sathiyaa, senior product manager, Real-time PCR Instrumentation, and Sejal Desai, senior product manager, OpenArray Consumables at Thermo, told IBO, this capability is unique, as it is the only qPCR system that offers several reaction formats on the same instrument. Mr. Sathiyaa and Ms. Desai explained that reduced hands-on time due to fewer workflow steps and automation, compared to traditional qPCR formats, reduce user-to-user variation.

OpenArray technology has evolved to include more formats, new assay types and new application areas. Analysis of mRNA and miRNA for disease biomarker association are among OpenArray’s most popular applications, according to Mr. Sathiyaa and Ms. Desai. The most popular genotyping applications for OpenArray technology is for personalized medicine and inherited diseases, as well as agricultural biotechnology. It is also used for gene expression applications in agricultural biotech.

Asked about the changes in end-user needs since the introduction of OpenArray technology, Mr. Sathiyaa and Ms. Desai told IBO, “Main end-user needs, such as lower cost per data point and ability to process numerous samples, has not changed since 2008. However, users are expecting simpler and more automated steps in the workflow.” This was addressed with the 12K Flex, according to them. They also told IBO that as the market has evolved, customers continue to want to reduce reagent costs and work with a large number of samples.

Asked about the future of the nanoliter qPCR market, Mr. Sathiyaa and Ms. Desai noted that efficiency and not lower volumes will be the focus. “As long as technologies offer reduced cost per sample, throughput and convenience, further miniaturization does not necessarily provide additional benefits to the end-user,” they said. “The emphasis will continue to remain on data quality and automation for users to increase efficiency in their day-to-day operations.” Rather, improved sensitivity will drive product development. “Future technologies in this space will most likely focus on improving sensitivity for certain applications, such as somatic mutation analysis which is currently limited due to reaction-volume constraints.” As for future growth areas, they listed marker-assisted selection and breeding in agricultural biotech, infectious disease and environmental applications.

One of the first companies to introduce a nanoliter qPCR format was Fluidigm, which launched its microfludic-based Dynamic Arrays in 2006. For Fluidigm, qPCR is one of many workflows for its IFC (Integrated Fluidic Circuit) technology, which utilizes chambers and valves to enable self-contained mixing and reactions. For genotyping or gene expression, the 96.96 Dynamic Array IFC, the highest-volume array, contains up to 9,608 reaction chambers. The Arrays are run on the company’s Biomark HD system, which also accommodates Arrays for end-point and digital PCR. One of three available IFC Controllers loads the Arrays, which are available preloaded or customized.

Although IFCs using qPCR have been integrated into the company’s sample preparation IFCs for NGS target enrichment and single-cell analysis, Fluidigm continues to offer Dynamic Arrays for gene expression and genotyping. “So for us, PCR is a component,” explained Marc Unger, PhD, senior vice president of R&D at Fluidigm. “We have a technological approach which provides a general architecture that allows us to change the design and layout of the devices, so we can use PCR in a lot of different device configurations.”

The ability to configure nanoliter qPCR to various applications is a differentiator of Fluidigm’s nanoliter qPCR capabilities, according to Dr. Unger. “So, for instance, we do PCR downstream of single-cell capture, lysis and reverse transcription on our C1 chips. So this is a functionality that we have as opposed to the only functionality that we have, which is pretty much the case for all the other folks who are doing nanoliter qPCR.”

Nanovolumes are especially suited to qPCR, explained Dr. Unger. “When conducting PCR-based experiments, smaller is better when it comes to amplification, and the reason for that is really simple: when you are doing PCR, you like the concentration of input molecules to be low in general.” He added, “I think our chambers are generally smaller, in the range of 5 nL to 15 nL, as opposed to some of the other folks who are doing nanoliter PCR using larger volumes, often sample fractions of a micro liter. When we want to have larger volumes, we can do so—5 nL to 400 nL, for example.”

Gene expression is one of multiple configurations of IFCs for nanoliter qPCR and is particularly well suited to single-cell gene expression due to the small amount of starting material. “This is part of what pulled us into the single cell biology field,” explained Mr. Unger. In addition, the use of the company’s C1 Single-Cell Auto Prep system to prepare the sample for single-cell gene expression with the Biomarker HD system creates an integrated workflow. Applications also include clinical research studies, such as patient stratification for clinical trials, according to Dr. Unger.

For genotyping applications, in which large numbers of samples are screened, IFC qPCR is also desirable. “For genotyping, a lot of people use the Biomark HD system to screen for inherited disease,” said Dr. Unger. “Really good applications for that typically have quite a lot of patients and using the Biomark HD, you can test a large number of loci at once.” Another application he mentioned was bone marrow screening, which is used to look for a donor match.

An evolution of Fluidigm’s technology for genotyping was this year’s introduction of the Juno system. It integrates preamplification, required for all IFC qPCR runs, onto the same chip as the assays. The 96.96 IFCs are currently available. Eventually, all of Fluidigm’s gene expression and genotyping IFCs will be available for use with the Juno. “This further automates chip preparation, leading to greater reproducibility,” according to Howard High, fellow, Corporate Communications and Press Relations, at Fluidigm.

Discussing the evolution of the market, Mr. Unger expected few fundamental technology changes but more refinement of the workflow. “The demands on ease of use and, of course, cost, I think continued to evolve. They are more changes in degree than changes in direction, if you will. . . . Mostly for customers in genotyping and gene expression, it has been changes in degree,” he noted, adding, “The big shifts have been [in] single-cell biology.”

Asked about the market’s future direction, Dr. Unger stated that it will continue to be influenced by sequencing, but that sequencing will not displace the need for qPCR. “Actually, I think NGS sequencing and PCR are going to coexist for a long time because sequencing is very capable but it also, to some degree, answers a whole bunch of questions you didn’t necessarily ask,” he noted. “There’s qPCR and genotyping assays out there that have been validated and that work great for what they’re being used for. And people are going to continue to use them. Cost wise and speed wise, often they are still the best answer.”

Applied markets will also be an area of future growth for nanoliter qPCR. Dr. Unger cited marker-assisted breeding in particular. Another area of growth is use by biorepositories.

“One of the applications for the genotyping chips, in particular, is biorepositories: to check the identity of the samples they are bringing in.”

WaferGen’s SmartChip nanoliter qPCR technology was launched in 2010 specifically targeting gene expression applications. Since then, the company has expanded the technology to NGS target enrichment and single-cell analysis applications, but continues to offer assays for gene expression, as well as genotyping. The metal alloy nanowell plate contains 5,184 wells, which is equivalent to 13 384-well plates. Custom or prevalidated panels are available, and samples are loaded using the SmartChip Multi-sample Nano Dispenser. In 2012, the company released the SmartChip MyDesign Real-Time PCR System, allowing end-users to prepare their own assays for SmartChips. As Yong Yi, vice president of Marketing at WaferGen, explained, “If you compare that to standard 96-well plates, you’re talking about being able to perform 54 96-well plates on a single chip. It has tremendous throughput and efficiency that it can provide.”

Advantages also include the technology’s flexibility. “There are a couple of levels of flexibility as well. So, number one is the types of assays you can run on the chip, but we also have great flexibility in the format of the chip as well.” Fourteen plate configurations are available. He added, “There may be other microfluidic or nanoscale type of systems, but the other nice thing about our chip is you can have flexible formats of different sample numbers and assay numbers on the chip and still get full utilization of the chip.” Preamplifcation is not a requirement in most cases.

Gene expression application of the SmartChip include validation of NGS, RNA-Seq, Chip-Seq and microarray studies, according to WaferGen literature. Discussing the SmartChip’s gene expression applications, Mr. Yi said, “I think a lot of the biomarker validation and discovery is applicable. You can also run disease-associated panels on this as well.” The company offers the Human MicroRNA Panel and the Human Oncology Gene Panel. The flexibility also allows the research to be taken downstream from basic research to clinical development. “A lot of human research, may be somewhat extending into clinical research type of applications.”

The SmartChip’s genotyping applications include validation of SNPs, as well as drug-response monitoring and rare mutation detection. Mr. Yi highlighted screening applications for human disease research as another genotyping application area. “There’s potential for sample quality control, sample ID–type applications within the plant and animal. There’s also genotyping that’s used commonly, for example, in seed companies to do breeding or QC-type applications.”

Mr. Yi also believes sequencing has influenced the evolution of the technology, not only for new applications, but the use of nanoliter qPCR for validation as the rate of discovery has increased. Customized assays have also been a key capability. “A lot of times people will embark on their own discovery and have identified a panel, and will want to port that onto a platform like ours to enable that kind of routine running. So that’s where the flexibility, or the sample and assay formats that we have, really become a nice feature.”