by Caitlin Smith
Real-time PCR
is a crucial step in many experiments today: to amplify and quantify DNA, to assess mRNA abundance, even to find small sequences changes. As more people come to depend on this technique, more people have worked to improve it—and this work has paid off, according to Barry Westfall, North America marketing manager for gene expression and function at QIAGEN:
“As new innovations develop in real-time chemistry and cycler technology, we are seeing the ease of use, sensitivity, throughput, and speed improve dramatically. Gene expression measurement is becoming much more routine.” However, there is nothing routine about the newest instruments and other tools on the market today.
Real-time PCR instruments
Several new instruments are helping to get more data from each real-time PCR experiment. Agilent Technologies
offers a new system, the Stratagene Mx3005P, which is an expansion of its well-respected workhorse, the Stratagene Mx3000P. While the latter offers dependable, solid performance for basic experiments, it is limited in the scope of more advanced applications, such as multiplexing. Researchers are increasingly taking advantage of multiplexing, in which multiple samples can be run together, with their readouts differentiated by fluorescent dyes with different emission wavelengths, for example. Agilent’s newer system offers greater multiplexing possibilities. “The Mx3005P QPCR System expands on the Mx3000P system by offering unmatched flexibility and capability to support even more real-time QPCR applications and chemistries,” says David Kerry, product manager for genomics at Stratagene. “The system features five customer-selected filters and a custom filter path feature to support detection of a broad range of dyes.”
Idaho Technology’s instruments are relied upon by lab researchers and first responders alike. Their new RAZOR®EX system is a portable real-time PCR machine “capable of detecting ten pathogens simultaneously with automated calling features, especially useful to military and medical first-responder groups,” says Cameron Gundry, scientist at Idaho Technology. “We have validated real-time PCR assays that relay the status of the sample, positive or negative, in under 40 minutes.” The new FilmArray™, aimed at clinicians, combines sample extraction and detection of up to 100 respiratory and sepsis-related pathogens in less than an hour. “In addition, the unique chemistry allows for species/strain differentiation utilizing post-PCR detection and requiring no sample manipulations,” says Gundry.
The method high resolution melting (HRM) analysis, in which a single base pair change can be detected by a change in fluorescence, is already letting researchers glean more information from their experiments than previously possible. In HRM, a fluorescent dye binds to double-stranded DNA until it is heated enough to denature, whereupon the dye falls off and no longer fluoresces brightly. The change in fluorescence with temperature is called a melting curve, shifts of which can indicate even a single point mutation. Idaho Technology’s new LightScanner® 32 “is the fastest practical PCR instrument in the world that also offers the highest resolution melting commercially obtainable,” says Gundry. “This instrument combines real-time three-channel detection chemistries with newer high-resolution melting dyes such as LCGreen® Plus.” Extracting more data from each experiment, while reducing effort and cost, is becoming a reality that many had hoped to see. “Multiplexing technology addresses this and so do chemistry solutions that are broadly useful, such as LCGreen® Plus for real-time PCR and end-point high-resolution melting analysis on improved instrumentation platforms,” says Gundry. “This economy of effort is a major step to realizing the dream of personalized medicine applications.”
“We are also seeing increasing adoption of high resolution melt (HRM) profiling,” says Bio-Rad’s Rachel Scott, product manager in the gene expression division. “Software, such as the soon-to-be-released Precision Melt Analysis software, provides access to these popular HRM applications, and also to the exciting and rapidly expanding field of epigenetic research.… For typical real-time PCR applications, we are seeing an ongoing push towards higher throughput and lower sample volumes.” Bio-Rad recently released their CFX384 Real-Time PCR Detection system, which is geared toward high-throughput, low-volume reactions in a 96-well format. “The CFX384 uses advanced optical design and thermal uniformity to provide reproducible results and worry-free operation, all from an instrument about one-third the size of current market offerings,” says Scott.
A cautionary note for HRM users, however, is expressed by Robertus van Miltenburg, global marketing director for qPCR and NAP systems at Roche Diagnostics. Roche Applied Science
has optimized HRM for performance on its LightCycler 480 system, which van Miltenburg says possesses thermal and optical properties that are well suited to “obtaining high quality raw data for further analysis.” He cautions that not all systems available can supply the same quality, which one may regret in later analysis. “Roche [has put] great effort in getting homogenous data by improving block heating and cooling, and by improving data capture with a high intensity xenon-lamp [that allows taking] CCD images from the entire block at once,” says van Miltenburg. “When using a block with lower quality, or using scanning systems for data capture, the raw data will require additional data normalization. For instruments with scanning optics, I have seen no satisfactory solutions to get the required resolution.”
An instrument that will help ameliorate this problem is QIAGEN’s new Rotor-Gene® Q cycler. With its novel centrifugal rotary design, sample tubes spin quickly in an air chamber, making the temperature uniform for all samples. This design also facilitates quick optical measurements as each tube passes the detection optics. Many Rotor-Gene Kits are available to ease your workflow, which QIAGEN has focused on for optimal experimental outcomes. “More companies, like QIAGEN, are looking at the entire research workflow to improve how the instruments and reagents perform together,” says Barry Westfall, North America marketing manager for gene expression and function at QIAGEN. “Rotor-Gene Kits for SYBR Green, probe, and multiplex are designed to enhance the precision, sensitivity, reproducibility, and speed in all real-time applications for the Rotor-Gene Q.”
Tools for your real-time experiments
Along with instruments, reagents and consumables are speeding PCR studies too. For example, SABiosciences
uses PCR array technology to make application-specific arrays for real-time PCR instruments, focusing on the gene expression of specific biological pathways or diseases. Jeffrey Hung, SABiosciences’ director of marketing, says that in addition to pathway PCR arrays, SABiosciences also has arrays for genome-wide microRNA expression analysis, methylation analysis, and real-time PCR-based ChIP analysis. Arrays like those offered by SABiosciences and others will provide more opportunities for high-throughput applications, which Hung says remain a challenge: “We will see breakthrough technologies that can make real-time PCR amenable for high volume applications such as drug screening. Multiplex, high-throughput sample prep and cost-per-assay need to be addressed to make high-throughput gene expression analysis possible.”
Applied Biosystems
also offers new tools for real-time PCR in expression assays. Their new TaqMan PreAmp Pools are preconfigured sets of reagents that help, for example, with RNA amplification from small biological samples. “The new TaqMan PreAmp Pools streamline the workflow for researchers working with small or limited sample materials, such as stem cells, laser-capture microdissected samples, needle biopsies, and paraffin-embedded tissues,” says Jon Sherlock, product manager of TaqMan Arrays and Express Plates for Applied Biosystems Genomic Assays. “Use of the PreAmp Pools with pre-plated [TaqMan Express Plates and the TaqMan Arrays] will allow researchers ease of handling, and affordable experiments on samples previously thought impossible to analyze. For example, researchers will be able to investigate entire biological pathways, gene signatures, or groups of disease genes, even from single cells.”
Indeed, progress in real-time PCR technology is driving the technique forward into other research areas, from disease pathways to microRNA expression.