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Jul 1, 2009 (Vol. 29, No. 13)

Getting the Most out of Quantitative PCR

Strategies and Solutions for Overcoming Common Problems in Laboratory Experiments

  • Strategies for Faster qPCR

    Click Image To Enlarge +
    White polypropylene plastics reflect the fluorescent signal back to the detectors more efficiently than clear plastics, resulting in significantly improved sensitivity and consistency between wells

    The demand for qPCR data is such that the ability to run faster assays, with higher throughput and without sacrificing efficiency and sensitivity, is of high value. Researchers often turn to instrumentation, reagents, and kits to increase speed, but tend to overlook the smaller details. Thermo Fisher Scientific has studied the optimization of speed in qPCR and its findings were reported at the meeting by Ian Kavanagh, Ph.D., R&D manager of genomics. He showed that small details can make a big difference in overall performance.

    One of the most significant ways that Thermo improved the speed and performance of assays was through the use of white plastic plates, Dr. Kavanagh said. Clear plastic is more commonly used, but white plastic reflects more signal back up to the detectors, giving a much greater sensitivity. A blue pigment in the master mix enhances the contrast between reagent and plastic and helps overcome the problem of not being able to clearly see the liquid in the wells.

    The other interesting result from Dr. Kavanagh’s presentation was optimizing fast cycling by investigating specific traits of the PCR primers, but not necessarily requiring the use of specialized fast-cycling instrumentation. “As long as you can maintain the same sensitivity and reproducibility, anyone is going to want to gather their data rapidly so they can move on to the next stage of research.”

    By the same token, most clinics and hospitals are eager to speed up qPCR-based diagnostic tests, but are limited by their equipment. Sundaresh Brahmasandra, Ph.D., vp of product development for Handylab, explained in his presentation how Handylab’s instrumentation overcomes common problems in a qPCR diagnostic assay system.

    Most of these systems are physically quite large, and occupy a great deal of space in the laboratory. They are also often closed systems, meaning that users are limited to the tests offered by the manufacturer. The systems typically operate at high throughput and run a large number of tests at one time. However, in practice, this means that many hospitals and laboratories save up patient samples until they have enough to justify a large run. This effectively slows down the diagnosis.

    Handylab has addressed these problems with the Jaguar benchtop system. The cost is competitive compared to the larger systems, Dr. Brahmasandra said. It can run up to 24 samples in two hours, he added, but the real advantage is that they do not have to be all the same test. In fact, they don’t have to be tests manufactured by Handylab at all. The user can run his or her own assays on the system, in whatever combination is necessary.

    “The underlying technology enables all of this to happen. We have a unique extraction chemistry and can prep from a variety of matrices,” said Dr. Brahmasandra. The other ingredient is a microfluidic vessel, allowing the reaction to be carried out in a 4 µL reaction volume, which permits faster cycling. The entire amplification and detection is complete in 20 to 25 minutes. “You can run 24 specimens, and they can literally be different specimen types used for targets with 24 different thermocycling profiles in neighboring locations...it’s like 24 mini-instruments within the system.”

    Improvements in efficiency and reductions in cost are a good sign that qPCR is a mature technology that is ready for prime time in the lab or clinic. There are many choices of methods and instrumentation, but the most powerful “upgrade” is in the knowledge and experience of the operator in setting up a well-designed, fully optimized assay.

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