Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
The advent of qPCR enabled researchers to ascertain how much of a specific DNA sequence is present in a sample, instead of simply whether it is present at all (as in endpoint PCR). That makes the technique useful for measuring RNA abundance, quantifying pathogen loads and validating microarray and sequencing data, among other applications.
First, though, you need to find the right qPCR instrument for your lab. Keeping a few variables in mind can make this a fairly straightforward process.
Ease of use
qPCR is performed using fluorescent indicators that emit light at intensities proportional to the copy number of the labeled DNA. The fluorescent indicator is generally one of two types, a fluorescent dye or a fluorescent probe. Fluorescent dyes, such as SYBR® Green, bind double-stranded DNA and are less expensive and easier to use than fluorescent probes. However, dyes cannot exclude off-target effects, and probes have the upper hand when it comes to multiplexing (with the exception of dye-based melt-curve analysis).
Holly Lin, product manager for real-time systems and software in the life science group at Bio-Rad Laboratories, emphasizes the need for the instrument and software to be intuitive. Lin suggests, "You should consider if it will be easy to get your experiments up and going as soon as you receive the instrument. Also, consider if the software is easy to use and if it will help you visualize and understand your data."
Bio-Rad real-time PCR systems are self-installable and have factory-calibrated optics. Their systems also incorporate easy-to-use software that aides in every step of real-time experiments. For further ease, Bio-Rad offers PrimePCR assays. "PrimePCR assays offer researchers guaranteed assay performance, saving researchers the time and hanssle of designing and validating their own primers. Furthermore, you can customize 96- and 384-well plates to quickly examine the expression of many genes in single single real-time PCR assay," Lin says.
Ease of use also can impact throughput, with improved technology making it easier to do more experiments faster and in less time. The Bio-Rad CFX384 supports a 384-well microplate format, for example. And Life Technologies’ QuantStudio™ 12K Flex Real-Time PCR System can produce up to 12,000 data points in one run, says Angelique Habis, senior market development manager in genetic analysis at Life Technologies.
“Utilizing [Life Technologies’] OpenArray™ technology, a single user can now easily complete most projects in days instead of weeks,” Habis adds. The system runs OpenArray® sample plates—up to four at once, which is the equivalent of 32 traditional, 384-well plates. Using a QuantStudio™ 12K Flex Automation Robot and several thermal cycler units, users can run more than 2 million data points per day, according to product literature.
PCR machines are also easier to use if they are located as close to your work space as possible. How about on your bench top. Think it won’t fit? The Thermo Scientific PikoReal™ Real-time qPCR system probably will. The PikoReal features a tiny footprint (about 30 x 23 cm) and energy-conserving operation, and provides significant energy savings compared to traditional qPCR instruments, according to Hanna Grano-Fabritius, business director for liquid handling consumables at Thermo Fisher Scientific. The instrument’s small size and low weight make it easy to transport to other labs for collaborations, or even outside for field work. The PikoReal is not lightweight in features, however; it can handle 24- or 96-well formats and perform four-target multiplexing. (Roche Applied Science's LightCycler® Nano instrument is comparably sized, measuring 24 x 27 cm.)
Dependability
Another key feature is the ability to produce high-quality, reproducible data. Lin suggests asking, “Will I get consistent results experiment after experiment? Will the quality of the instrument give me confidence in my results?” Talking to colleagues who have used a variety of systems can help you identify which instrument might fit your lab situation best.
Specific points are worth considering when it comes to the quality of data generated on a qPCR system—thermal uniformity, for instance. Non-uniform temperatures across wells can create data inconsistencies, because small temperature differences can affect both primer annealing and fluorescent signals, says Carola Schade, director of life science instruments at QIAGEN.
To support temperature uniformity across the heating block, QIAGEN’s Rotor-Gene Q qPCR instrument uses what the company calls a “unique centrifugal rotary design” and a circulating-air-based temperature-regulation system. According to Schade, this heating and cooling system outperforms Peltier-based systems at preventing temperature fluctuations and “hot spots” at the edges and corners of the block.
Flexibility
As with many lab instruments, a qPCR platform should be flexible enough to adapt to the evolving needs of the lab. For instance, you may need the instrument to be compatible with automation, says Lin.
Bio-Rad’s CFX384 fits the bill. So, too, does Life Technologies’ QuantStudio line. The automation-accessible QuantStudio™ 7 Flex Real-Time PCR System lets researchers use different dyes or sample formats, while the QuantStudio™ 3D Digital PCR System allows for “absolute target quantification without the use of a reference or standard curve,” says Habis.
Some qPCR platforms are flexible in the applications they support. For example, Roche Applied Science’s new LightCycler 96 System is a qPCR system with 96-well capacity that supports absolute and relative quantification, melting curve analysis, endpoint genotyping and high-resolution melting and qualitative-detection applications, according to Julie Desnoyers, international product manager for NAP/qPCR at Roche Molecular Systems.
Desnoyers says that the most important features to look for in a qPCR system are its ability to maintain temperature homogeneity across wells, and to excite and capture data from all wells equally. “The most crucial aspect is to rely on an accurate system that generates data you can have confidence in and that produces publication-ready results,” she says.
Ultimately, though, an instrument is more than just a collection of specs. Make sure the system works for your application, but don’t neglect service considerations. Says Habis, “It is important to know you have support behind that instrument, such as easily accessible service engineers that are well trained, as well as applications support and training that [you may] need for today and in the future.” After all, a qPCR instrument isn’t inexpensive, and you’re going to be using it for years to come.
Image: Life Technologies' QuantStudio 6 Flex Real-Time PCR System.