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Real Savvy For Your Real-Time Purchase
Buying Tips
Oct 6 '04
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Introduction |
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 Perhaps the most difficult thing about real-time thermal cyclers is deciding on which one to purchase. While today’s researchers are blessed with such enabling technology, they are also tasked with the duty of sifting through marketing brochures filled with alluring claims, gathering opinions from colleagues and making realistic assessments about the lab’s current and future needs. Then, unlike a kid in a candy store who may buy several treats, you must select the one device for which you will pay anywhere from $15,000 to $100,000.
If you like to use popularity as a gauge of whether or not to purchase a real-time thermal cycler, consider devices made by Applied Biosystems, Roche Applied Sciences and BioRad, which accounted for 85% of all real-time thermal cyclers sold in 2002, according to a Frost & Sullivan report. (1) But, if you don’t care what others are buying, finding the right one requires looking at several features and capabilities.
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Consider Throughput |
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| First, think about how often you and others in the lab will be using the device. “If you’re only going to use it once in a while or if you’ll be using it to check something that you already know, then it’s not important to buy the high-end instruments,” said David Ginzinger, director of the genome analysis core facility for the comprehensive cancer center at the University of California, San Francisco. Labs that require higher throughput, such as those looking for drug targets or disease markers, may require thermal cyclers that can accommodate 384-well plates and promise faster cycling times.
Your high-throughput lab may want to go a step further and buy automated instruments that can process samples and prepare the resulting nucleic acids for PCR — all at the touch of a button. With Cepheid’s soon-to-be launched GeneExpert, you can get amplified nucleic acids a half hour after you load in a cartridge of raw sample. The device lyses cells and purifies and amplifies the nucleic acids. Roche’s MagnaPure is also an automated nucleic acid purifier that includes PCR preparation for the company’s real-time thermal cycler, LightCycler.
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Flexibility With Add-Ons |
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| Because instrument developers know your needs change over time, they have designed devices to come with optional attachments and modules, such as blocks that handle different types of plates of reaction vessels. Some companies offer optical attachments to enable basic thermal cyclers to also perform real-time PCR. BioRad offers optical modules for its MyCycler to upgrade it to real-time. Cepheid’s SmartCycler II is a good option for labs that have will only do a minimal amount of real-time PCR at first but may do more in the future. The basic package comes with one block that can process 16 independent reactions. Then, as your needs increase, you can hook up to six blocks to the one cycler.
“It works so that it’s upgradeable and low cost,” said Kurt Petersen, president and chief technical officer of Cepheid. He also pointed out the convenience of being able starting the 16 different amplifications at different times and with different parameters. Calling it “random-access capability,” he explained that “it’s good for cash-strapped universities.”
“This is a huge value to researchers,” Petersen said. “That means a graduate student can start [amplifying] a few samples, then 10 minutes later, someone else can start their few samples independently.”
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Multiplex Mania |
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| Multiplexing seems to be all the buzz these days. And real-time thermal cyclers have not been immune to the craze. This capability is available in high-end instruments. With more than one optical channel, the device can track the amplification of more than one type of oligonucleotide. The newer 2.0 version of the LightCycler comes with six optical channels while its predecessor, the 1.2 version, has three channels. Cepheid’s SmartCycler II has four channels. Whereas LightCycler 2.0 costs $57,500, you can purchase Stratagene’s Mx3000P, which is capable of four-color multiplexing, for just under $25,000.
But multiplexing isn’t for everyone. The complicated reaction set-up “is more effort than it’s worth,” says Ginzinger, who has tested most cyclers on the market. He suggests that people use the high-throughput devices that allow for 384-well plates, instead of trying to increase efficiency with multiplexing.
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Software Are Not Created Equal |
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| Whether you decide to purchase a multiplex-enabled device or not, your next step should be to examine the accompanying software. Is it user-friendly? Does it do the types of analyses that you would require? What kinds of output formats does it allow? While some software packages may be user-friendly, they may be too simplistic to do more complicated analyses. Older packages require you to perform further analysis and manipulation after off-loading data from the device. Software in the newer thermal cyclers is programmed to do all the work.
Aside from offering a selection of basic reaction parameters, some software may offer features that improve efficiency and the accuracy of your results. “Quantitative PCR takes a few copies and amplifies it billions of times, so if you’re off by a copy number you’re results could be off by magnitudes,” said William Demyan, director of applications and technical services for Roche Applied Sciences.
To maximize precision, look for software offerings that help with primer and probe design, assay design and development, multiplex assay design, adjustment for background noise and data normalization that calculates data relative to a baseline signal in each reaction vessel.
“It’s important to talk to people who are using it for a similar application,” Ginzinger said.
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Inconsistency Realities |
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| Even though software can make certain corrections, it’s still a good idea to make sure you purchase a device that minimizes the need for adjustments in the calculation. Ask about the well-to-well variability of the device, Ginzinger said. This could be due to minute differences in the fiber optic cables that receive and transmit signals or subtle variations in the lenses or in the parts of the reaction vessel that stands in the path of signal transmission.
Ginzinger has performed uniformity tests on devices from most companies. He places the same samples and reagents in each well and runs it through real-time amplification. Many times, the wells have different results. “Just looking from well to well, it’s amazing how different each instrument is,” he said.
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To Kit Or Not To Kit |
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| Purchasing reagent kits is not necessarily the solution to dealing with the problems of variability. While the sales force may tout the superior quality control of kits, solutions that you mix up in the lab can offer comparable results.
“Kits aren’t immune to variability,” Ginzinger said. He suggested buying kits for labs that will only occasionally perform real-time PCR. But if the protocol is a major part of your research, the cost of kits may be prohibitive.
For the best results, though, make sure to thoroughly formulate and assess your experimental set-up and data analyses. “Oftentimes people look at real-time PCR as a quick and dirty method and expect good results,” he said. “You won’t.”
And, “If you’re careful with running our experiments, you can get good data with pretty much any machine.”
References:
1. J Wilhelm and A Pingoud, “Real-Time Polymerase Chain Reaction,” ChemBioChem, 4:1120-1128, 2003. |
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Laura Lane
Contributing Editor
Related Product Links:
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qPCR Master Mixes
Quantitative PCR Labels
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Real-Time PCR Thermal Cyclers / Thermocyclers
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