by Catherine Shaffer
Don't have time to pour your own agarose gel, make your own buffers, and wait more than an hour for the results? You're in luck, because nucleic acid electrophoresis is getting easier every day. Using precast gels, all-in-one kits, and even automation, you can make a time-consuming, tedious task as simple as microwaving a bag of popcorn—and get better results than your grandfather did when he was pouring his own gels back in 1990. And although agarose and polyacrylamide gel electrophoresis are reliable, rock-solid techniques that have been in use for decades, there are still some surprising innovations to be made by thinking outside the gel box.
Bio-Rad offers one of the broadest product lines for nucleic acid electrophoresis, including systems for horizontal and vertical gel electrophoresis, the CHEF system for pulsed field electrophoresis, nucleic acid sequencing, DCode for mutation analysis, blotting systems, and a full selection of reagents, stains, and buffers. All of their products for conventional nucleic acid electrophoresis have recently been upgraded and optimized to be faster and more convenient. “We try to make it as painless as possible,” says Veronika Kortisova Descamps, MS, product manager for the gene expression division. “The researcher doesn't have to expend any effort thinking which way they should put their gel inside the apparatus. Even repair is made easy: it takes about five seconds to remove and replace the electrodes in our systems should they break.”
Besides traditional nucleic acid electrophoresis technologies, researchers are looking for the next wave of solutions that won't take a big chunk of time out of the work day, or consume too much sample. To that end, Bio-Rad also offers an automated, microfluidic-based electrophoresis solution for RNA and DNA analysis (as well as protein) with their Experion™ Automated Electrophoresis System. Different chips are available, depending on the biomolecule to be separated, and require very little sample for analysis. The RNA chip, for example, can accommodate as little as 5 ng (Experion RNA StdSens Analysis Kit) of RNA, conserving precious sample for downstream applications such as RT-qPCR. New guidelines for qPCR called MIQE1, which emphasize the importance of RNA quality and integrity, are percolating through the scientific community. The Experion system helps researchers meet MIQE guidelines when performing their experiments.2
“When considering RNA quality, many people are only concerned with purity. However, the physical integrity—whether the molecule is degraded or not—is an important factor in the success of subsequent experiments. Experion's RQI (RNA Quality Indicator) tool provides a standardizable measure of the quality of RNA on a scale of one to ten”, says Mary Grace Brubacher, M.Sc., marketing manager for the laboratory separations division at Bio-Rad.
Lonza offers a complete set of solutions for electrophoresis of nucleic acids and proteins, including agarose powder, precast agarose gels, precast SDS and native PAGE gels, standards, stains, and buffers. Their products are tailored to cover a broad range of applications and needs, from quick and simple flash gels, to products that support very technically advanced applications such as sequencing and qPCR.
Super fast gel systems like Lonza's FlashGel® are one of the hottest trends in the industry. The idea behind it is that a small, quick gel analysis can give you a useful snap shot of your DNA or RNA before you move on to your next steps. The FlashGel system is a palm-sized combination electrophoresis box and transluminator unit. It uses premade cassettes that contain precast, prestained gels. The gel runs at 275 volts, more than twice the recommended 110 volts for a standard agarose gel. “It runs so fast,” says Mary Riley, “that you can literally watch the bands migrate ... for most fragments you see clear separation of your bands within five minutes or less. For some fragments it's as little as two minutes.” A popular use for the Flash Gel is to analyze a PCR product quickly to see if the reaction was successful. Another, lesser-known benefit of the self-contained FlashGel system is that it minimizes exposure to visual and UV light, which can damage and degrade nucleic acids.
In recent years, Lonza has tracked an increase in customers doing electrophoresis to support next generation sequencing, qPCR, and genotyping. Agarose gel electrophoresis is such a versatile method that it shows up in virtually every lab, and Lonza brands such as SeaKem, NuSieve, and Metaphor have become staples of the industry. "Because of its unparalleled purity, our agarose is also used for some interesting non-electrophoresis applications, such as delivery mechanisms for pharmaceutical applications," says Riley.
Even with a technology as old and familiar as agarose gel electrophoresis, it is still possible to come up with a new idea that changes the paradigm completely. This is the case with Faster Better Media (FBM). The company is organized around a single, simple idea—a low conductive buffer that lets your gel run at high voltages, and thus you get high resolution separation of your DNA bands on your gel in a fraction of the time it takes to run a conventional gel. Using FBM's LB buffer, it is possible to crank up the voltage on your gel box and complete the entire analysis in fifteen minutes or less.
After so many years of babysitting gels and fretting about overheating, it's difficult for many scientists to make the transition. “We get sort of funny emails from people that say, 'I got great results when I ran it at 250 volts. Our thing goes to 300, but I'm nervous to run it that high.' I write back, 'Go for it!' and they get excited and they do it and it works!” says Jon Brody, PhD, co-owner and co-inventor at`1 Faster Better Media.
The heat problem in conventional running buffer derives from the tris component of the buffer. Tris buffer has high conductance, and generates a high current. The high current increases the heat and conductance of the buffer, which increases the current further. The patent-issued, lithium-based buffer from Faster Better Media is low conductance and does not generate this feedback loop. The use of tris borate EDTA (TBE) or tris acetate EDTA (TAE) buffers for DNA and RNA electrophoresis is based on tradition, not optimal utility. Until recently, it was not questioned.
Nucleic acid electrophoresis is a very well-known, common technique. However, because people are using nucleic acids in new ways, such as in qPCR and next generation sequencing experiments, it's worth taking another look at the old agarose gel, to see if you are still getting the most out of your setup, your buffers, and your stains.
References:
1Bustin, SA, et al., "The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments." Clin Chem, 55, 611–22, 2009.
2Taylor, S, et al., "A practical approach to RT-qPCR publishing data that conform to the MIQE guidelines." Bio-Rad Bulletin #5859, 2009.