by Caitlin Smith
There is a wide range of applications, including genotyping, SNP analysis, drug target validation, and quantitative gene expression analysis, which require quantification of the amount of DNA in a starting sample. Increasingly, researchers are utilizing real-time PCR, or quantitative PCR (qPCR), for such quantitative measurements. Real-time PCR derives its name from the fact that it monitors PCR amplification as it occurs. qPCR can also be combined with reverse transcription (RT-qPCR) to quantify the amount of RNA in a starting sample. No matter which real-time system you choose, the instrument will include a thermal cycler to raise and lower the sample temperatures during cycling, and software for protocol set up and data analysis. On the reagent side, there are several options for DNA detection; some of the most common are SYBR® Green (double-stranded DNA binding dye), Invitrogen’s TaqMan® probes (fluorogenic sequence-specific probes), and dual hybridization probes, which use two probes for added specificity. To save time and expense, many real-time PCR reactions can be done in multiplex. If you are looking for a real-time system, here are some considerations and recent advances that can help you to make an informed choice.
Small and mighty
Space is always at a premium in busy labs, but now you can find powerful instruments in a compact size. In June, Thermo Fisher Scientific is releasing “a new personalized real-time PCR instrument for the individual researcher,” says Hanna Granö-Fabritius, market and product line manager in sample preparation and analysis at Thermo Fisher Scientific (however, note that it will not be available in Canada, Brazil, UK, Germany, Austria, Switzerland, Italy, Spain, France, Belgium, the Netherlands, Luxemburg, Denmark and Sweden until later in 2012). The new PikoReal Real-Time PCR System will be available in 24-well and 96-well configurations, with a small footprint of only 30 cm x 23 cm. “It still offers all the features of a standard qPCR instrument, including exceptional performance, fast cycling times, HRM [high resolution melt] and up to 4-plex multiplexing capabilities,” says Granö-Fabritius. The unit is based on the smaller Piko PCR Plates; these are 1/4 of a standard PCR plate and still compatible with standard multichannel pipettes and liquid handling systems, yet they save energy and reduce plastic waste.
The PikoReal can also help eliminate waiting times for instruments. “Many researchers working with real-time PCR applications still share instrumentation with multiple users and may have to reserve time beforehand to get access to a unit,” says Granö-Fabritius. “I think many researchers would benefit from having more personalized instrumentation, easily accessible and ready for use any time.” Personal instruments also tend to be more user-friendly, something that Granö-Fabritius recommends looking for. “On the whole, I think there is still a lot of room for improvement in the usability of real-time cyclers,” she says. “This applies especially for control and data reduction software, which really do not reach the level of ease-of-use as for many other types of laboratory instrumentation.”
Illumina also aims to make real-time PCR more accessible to individual researchers with their compact Eco Real-Time PCR System. This instrument can run four-color multiplex applications, and can support other real-time applications such as absolute quantification using a standard curve, relative quantification, allelic discrimination by end-point fluorescence, and HRM analysis.
Eppendorf's Mastercycler® ep realplex is another compact instrument that saves lab space. It also aims to save time with fast temperature ramping and short cycling times. It has an optical system and lid that include technology to “prevent premature heating of the samples and the associated amplification of nonspecific PCR products during the heating phase of the heated lid,” says Jaimie McLaughlin, product manager in PCR, detection and cell technology at Eppendorf. “The optical lid is also equipped with very sensitive photomultipliers of the latest generation. Photomultiplier tubes show greatly increased sensitivity of your emission wavelengths. The fluorescent dyes are excited by 96 individual LEDs, which have substantially longer lifespan in contrast to other light sources.” McLaughlin also notes the importance of sealing reaction vessels tightly. “In addition to the prevention of contamination, effective protection against evaporation must be considered,” she says. “Therefore, we recommend that sample plates be heat-sealed with a suitable heat-sealing film.”
New twists on traditional technique
As real-time PCR use became more widespread, thermal cycler vendors developed their products to have more features than ever, with many features specialized for particular applications. For example, Cepheid’s SmartCycler System is extremely flexible, with 96 individually programmable wells. If needed, you can run a different protocol in each well simultaneously. Or, two or more lab members can run concurrent experiments to save time. The SmartCycler System software optically monitors each individual well as the fluorescent signals develop with amplification. The system also allows 4-channel multiplexing assays.
Idaho Technology’s LightScanner® 32 (LS32) is a general, all-purpose real-time PCR machine with 3-color multiplexing capabilities, driven by a rapid air thermocycling system. It is particularly suitable for HRM. Cameron Gundry, Idaho’s associate marketing manager in the life sciences division, says “because of its superior melting data quality, several options open up for more efficient use of laboratory resources. For example, there is no need for expensive fluorogenic probes for genotyping. We suggest using unlabeled probes or amplicon genotyping with high resolution melting. During the high resolution melt protocol, there is no movement of the sample, and Roche® glass capillaries are used, providing the highest optical properties, and resulting in lowest noise.”
Gundry says that most LS32 customers are doing high-quality qPCR at a low to moderate throughput, as well as melting protocols for genotyping and gene scanning. To researchers choosing a real-time PCR system, Gundry recommends “honestly assessing your throughput requirements and realizing that there is a tradeoff between quality and quantity of samples run.” If you do require higher throughput with HRM, their 96- or 384-well LightScanner plate-based Hi-Res Melting® systems are compatible with most thermal cyclers. “For instance, the precise gradient and fast and accurate cycling conditions of Eppendorf’s realplex system allow for an unbeatable combination of real-time and melting data quality in the 96-well format.”
Qiagen’s twist on real-time PCR machines involves replacing the traditional heating block with a chamber in which samples are spun in moving air to control their temperature. The Rotor-Gene® Q measures signals as the tubes rotate past the excitation and detection optics. “This removes much of the variation that occurs in block-based systems,” says Marc Egelhofer, manager of marketing and public relations at Qiagen. “This is extremely important in challenging applications such as HRM analysis.” Qiagen is expanding its application kits for the Rotor-Gene Q, which include gene expression analysis, genotyping, pathogen detection, food safety testing, and forensics. Their recent release, the QuantiFast® Pathogen +IC kits, are designed to detect pathogen nucleic acids using sequence-specific probes (and also work on other real-time thermal cyclers). “To enable high process safety through correct interpretation of negative detection results, each kit contains reagents for duplex real-time detection of a user-defined pathogen target, such as viruses, bacteria or fungi delivered with a universal internal control,” says Egelhofer.
In choosing a real-time thermal cycler, Egelhofer recommends considering how well the system can grow with the lab, and with future experiments and applications. “The trend is to move towards integrated workflows, and Qiagen is meeting that demand with the QIAsymphony RGQ system,” he says. “It combines the sample preparation of the QIAsymphony SP, the assay setup of the QIAsymphony AS, and the real-time PCR cycler Rotor-Gene Q. This system allows you to fully automate a variety of assays on a single platform and comes with a broad range of applications.”
Higher throughput still in demand
Bio-Rad’s CFX96 and CFX384 optical reaction modules transform their C1000 thermal cycler into a powerful, multiplexing real-time PCR detection instrument. These devices are prime candidates for the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) application, sponsored by Bio-Rad Laboratories. “The MIQE guidelines provide a standard for qPCR research that will enhance a publication’s accuracy, transparency, and reproducibility,” says Rachel Scott, senior product manager of the gene expression division at Bio-Rad. “Developed by real-time PCR experts Michael Pfaffl, a professor at Techniche Universität München, and Afif Abdel Nour, an associate professor at LaSalle Beauvais, the MIQE qPCR app provides researchers with the checklists and resources needed to ensure MIQE compliance for their qPCR experiments in a convenient hand-held format.”
Applied Biosystems of Life Technologies’ new instrument, the ViiA™ 7 system, takes advantage of hundreds of customizable TaqMan® Array Microfluidic Cards, which contain preloaded TaqMan® Gene Expression or MicroRNA Assays. The Cards give you the same throughput as a 384-well plate, but without the need for plates, robotics, or complex pipetting protocols.
Roche has a new instrument for high-throughput work called the LightCycler 1536, “which is an open well 1536-well qPCR platform that allows highly reproducible data generation in sub-microliter reactions,” says Larson Maniford, marketing manager at Roche Applied Science. “The LightCycler 1536 instrument is built on a lot of the same hardware innovation that the LightCycler 480 had.” So what makes the LightCycler 1536 different from the 480? “There are other ultra-HT qPCR instruments on the market,” continues Manifold. “What makes the 1536 different is that it’s an open platform. It’s a similar technology to a 96 or 384 well plate that’s scaled up to 1536 wells.” The models also differ in sample volume: using the 384-well block, sample sizes can be as small as 5µl using the LightCycler 480 buffer, and as small as 3µl using the LightCycler 1536 buffer.
Manifold notes another key feature of the LightCyclers is the optics system – specifically the relatively long, 4-foot focal length. “This allows the instrument to minimize or marginalize edge effect on the plates,” says Manifold. “A lot of manufacturers need to compensate for this edge effect by adding, for example, a passive reference dye, so they can normalize the data within the software of the instrument. What this longer focal length allows us to do is to minimize that effect so we don’t need any kind of passive reference dye.”
A new take on PCR enzymes
PCR enzymes are evolving along with thermal cyclers. For example, Agilent Technologies recently released their Brilliant III Ultra-Fast reagents for real-time PCR. “Unlike other qPCR master mixes which utilize Taq, Agilent's Brilliant III Ultra-fast qPCR and RT-qPCR products incorporate an enzyme engineered specifically for sensitivity and speed,” says Laura Mason, global product manager, qPCR portfolio at Agilent Technologies. “In addition, the enzyme has shown to be more tolerant to inhibitors present in the reaction than Taq. Brilliant III Ultra-Fast also utilizes a novel hotstart technology that has the specificity of a chemical hotstart, but the speed of an antibody hotstart technique. Together these improvements deliver greater sensitivity, specificity, and speed from qPCR experiments.
Roche Applied Science is also offering a new qPCR enzyme – in this case, a modified Taq polymerase. “This enzyme does not require a hot start on the LightCycler 1536, so the approximate run times are between 40 – 50 minutes,” says Joe Donnenhoffer, technical service scientist at Roche Applied Science. “It is still Taq polymerase, but it’s an adaptor based technology.” Most hot-start enzymes require you to heat the enzyme for a certain length of time before it becomes active. “With our new enzyme, it’s inactive until you start your first cycle, and then it rapidly activates,” says John Ogden, director of technical support and applications at Roche Applied Science. “And then as you go through the various cycles it activates more enzyme. You’re protecting some of the enzyme and releasing it as you go, so you get this continuous release of enzyme activity, so that you don’t release it all up front and have it peter off as you go through the process.”
Making real-time PCR faster, with smaller volumes and more reactions – Ogden believes that we are reaching a biological barrier. “We’ve gotten to the point where now the limiting factor sometimes is just how fast the biology can take place,” he says. “So everyone’s trying to get to miniaturization, and multiplexing, and getting faster, and we’re getting to the point where that’s much more complicated.” If we’ve have reached the limits of these methods, what’s next?
One possibility, according to Ogden, Donnenhoffer, and Manifold, is a shift to a new type of analysis. “The shift might be, for instance, to isothermal polymerase reactions, or isothermal amplification,” says Ogden. “Rather than having to do the cycling of the heat up and down, in order to allow the DNA and the primers to melt, and then to reanneal, and then to extend, can we figure out how to do that basically in a single environment with everything there so that that’s continually in flux.” Are there things other than heat that could cause primers and the newly-made strands to separate and allow primers to bind again without having to cycle? “People are working on this so that now amplification can be 10 minutes instead of 40 minutes, because they’re getting away from having to cycle,” he says. “Because when we cycle, we are at the limit of how fast we can heat and cool the metal that we’ve got our tubes in. If we can get rid of that, maybe we can make it even quicker.”
The image at the top of this article is Bio-Rad's CFX384 Real-Time PCR Detection System.