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.
A new PCR machine is a big commitment, both financially and in terms of workload—few pieces of equipment are used as heavily. As you hunt for the perfect hardware, consider these five variables.
Endpoint, quantitative or digital?
Today’s PCR machines typically fall into three categories: standard, “endpoint” PCR; quantitative real-time PCR (qPCR); and digital PCR.
Standard PCR machines are used mainly to create an abundance of a nucleic acid sequence that will be used subsequently, for example in sequencing, cloning or simply to check some reaction on a gel.
qPCR, in which a target sequence is quantified in real-time rather than at the end of the reaction, frequently is used to measure the abundance of a biomolecule (such as a polymorphism or mRNA) rather than produce an end product. “With qPCR, [researchers] don’t care a lot about what happens to the PCR product when they’re done,” says Joe Donnenhoffer, lead technical service scientist at Roche. “They’re looking at questions like: What [did] I have when I started? Is my mutation present? … [and] How much target did I have when I started my reaction?” .
Digital PCR is also quantitative, but not real-time. In this case, the reactions occur in a large number of tiny partitions. The partitions are small enough that each one is likely to contain either 0 or 1 molecules of DNA; by counting the positives, researchers can obtain a true quantitative measurement of DNA that is more sensitive than qPCR and requires no standard curve.
Researchers generally know whether they want a standard or quantitative PCR machine, of course. But when it comes to choosing between qPCR and digital PCR, researchers sometimes need help, says Viresh Patel, senior marketing manager, Digital Biology Center, Bio-Rad Laboratories. Usually the choice comes down to the researchers’ intended applications and the questions they hope to answer. “When we hear things like copy-number analysis or mutation detection, that’s typically where digital PCR will provide a much better answer, a more unambiguous answer,” says Patel. But for high-throughput applications, qPCR usually is more practical (see below).
Sample format
Most users run PCR reactions in a 96-well format, usually with 0.2-ml tubes. Other formats are available for higher throughputs and different sample volumes, such as 0.5-ml tubes. Bibby Scientific’s Prime instruments “allow different sized PCR tubes to be inserted into the same block, which is useful when different people in the same laboratory are performing different PCR tests,” says Jim Bratherton, Techne product manager at Bibby Scientific.
Although some PCR machines have a fixed sample-block format, others are interchangeable. In deciding what to purchase, consider future plans, says Nicole Quackenbush, senior product manager at Streck. “Researchers should avoid the ‘that's how we've always done it’ mentality and explore systems outside of a standard 96-well block format,” says Quackenbush.
Some PCR machines allow the sample block to be exchanged for two or more smaller blocks. Such modular systems—which function almost like multiple thermal cyclers—can increase efficiency. Bibby Scientific’s dual-block design enables researchers to run different protocols for each of two or three blocks at once. Likewise, Streck’s PCR instruments contain four thermal modules, allowing four protocols to operate independently. “This design provides flexibility and significantly decreases optimization time, which means quicker time to results and discovery,” says Quackenbush.
An alternative to block-based PCR machines is a rotor-based system, which controls temperature by heating the surrounding air. “The rotor-based system is essentially like an oven,” explains Carola Schade, director and head of the instruments business in life sciences at Qiagen, whose Rotor-Gene Q qPCR system is rotor-based. “This format allows for significantly faster heating and cooling cycles as well as more precision with heating to specific temperatures.”
Throughput
For lower sample throughput, says Schmidt, “the most popular configuration is the standard 96-well, 0.2-ml block format.” But higher throughput, 384- or 1,536-well formats also are available.
Roche’s Donnenhoffer encourages customers to consider both their current throughput needs as well as their likely needs five to 10 years out when selecting an instrument. Roche’s LightCycler® 1536 can process 1,536 samples simultaneously in about an hour using a conventional-format qPCR plate. Bibby Scientific’s Prime instruments run up to 384 samples individually, but can also be hooked together for even greater throughput. “Some machines, such as the Prime Elite, [are] able to link three satellites, or slave units, via USB cables to the main thermal cycler in order to allow up to 1,536 samples to be run simultaneously,” says Bratherton.
Other systems, such as Bio-Rad’s CFX384 Touch™ Real-Time PCR Detection System, use a 384-well format but add the advantage of automation and robotics to churn through the plates. “With 384-well systems plus automation, you could run 20 plates in a day,” says Patel.
In general, digital PCR has more limited throughput, however. Bio-Rad’s QX200™ Droplet Digital™ PCR System, for instance, has 96 wells. The method’s key advantages are greater sensitivity and discrimination, says Patel. For high-throughput work, consider qPCR instead.
Portability and footprint
Some PCR machines are designed for portability, enabling researchers, investigators and military personnel to test for particular genetic materials in the field or outside the lab. But in most cases the instruments work on crowded lab benchtops, meaning a small footprint is a plus.
But they can only get so small, says Bratherton. PCR instruments require certain common components for proper functioning, such as heating elements, a power supply and a touch screen. “For this reason, high-performance thermal cyclers have a certain minimum size. Miniaturization will always be limited to some extent.”
User experience
Ease of use is essential for smooth operation and to minimize errors. Anyone who is new to PCR will likely find today’s touch-screen instruments equally easy to use, says Donnenhoffer. But advanced users may find it difficult to switch from one brand of PCR machine to another, because they already are used to a particular interface, he says.
“We believe that the software interface is critical for the usability of the instrument and the overall user experience,” says Schade, especially in diagnostic labs. Also enhancing the user experience is remote access, which enables users to adjust protocols and view preliminary results, all from a smartphone or tablet.
Ultimately, says Donnenhoffer, experienced researchers typically focus on two key parameters: speed and accuracy. With beginners, though, speed is secondary. “At the end of the day,” he says, “you hang your hat on accuracy.”
Correction (10/16/14): A comment in the "Throughput" section was incorrectly attributed to Peter Skirpstunas at Bio-Rad Laboratories; the comment was made by Bio-Rad's Viresh Patel.