Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
Let’s face it, gel electrophoresis is a pain. It requires making buffers, pouring gels, loading wells, following their migration, imaging and documenting the results and crunching the data—then dealing with the toxic waste—all to assess the relative migration of some macromolecules. Pre-cast gels and pre-made buffers have made things a little easier, but many researchers would still welcome more automation in their nucleic acid (and protein) analysis workflow. Now, thanks to automated gel-electrophoresis systems, they can get it.
Advantages of automation
Several commercial systems are capable of automating some or all the steps required to run a gel. As with slab-gel electrophoresis, these systems take advantage of the macromolecules’ propensity to differentially migrate through a lattice meshwork gel in response to an electrical field. And to a large extent, automated electrophoresis does what slab gels do—size determination, quantification, looking for integrity/impurities and DNA-fragment analysis—says Knut Wintergerst, marketing and support manager at Agilent Technologies.
Yet for the most part these meshworks are contained within narrow enclosures such as glass or polymer capillary tubes or microfluidic channels. Macromolecules pick up a fluorescent dye as they traverse the gel, enabling the instrument’s optical system to detect them as they pass by. This lets researchers visualize separations on the fly and immediately, automatically document and digitally archive them. 21 CFR Part 11 compliance packages are often available.
Such instruments also offer added advantages in hands-on time, overall speed, ease of use, reproducibility and/or sample requirement—in some cases as little as 1 μl of a 5 pg/μl DNA solution is enough.
In addition, instruments such as the Agilent 2100 Bioanalyzer allow for a measure of RNA-integrity assessment that cannot be achieved using slab gels, says Wintergerst. “RNA is a very fragile molecule,” he says. “Before you transcribe RNA to cDNA, you want to make sure it’s [of] good quality.” The 2100 Bioanalyzer software applies a neural-network algorithm to a variety of parameters extracted from the electropherogram (the digital trace generated by the instrument) to assess sample integrity, assigning it an RNA Integrity Rating (RIN) score between 1 and 10, with 10 implying a high-quality sample and 1 or 2 indicating highly degraded RNA. RIN has become an industry standard; other RNA-integrity scoring algorithms have since been developed, as well, and great pains generally are taken to demonstrate their correlation with RIN (for example, Bio-Rad Laboratories’ Experion™ RNA Quality Indicator, RQI).
Automation
Some automated electrophoretic systems, like the Bioanalyzer and Experion, can handle a dozen or fewer samples at a time and essentially automate from the same point at which a slab gel would be loaded. For these, a gel (sieving polymer)/dye mixture is forced under pressure into the channels of a microfluidic chip, and then samples are pipetted manually into the wells. After voltage is applied, the samples are transported through the chip’s architecture. Kits—which include gels, reagents and chips with different internal configurations—are designed for different size ranges of DNA, standard or high resolution and even protein separations.
Other instruments are built on a multiplexed capillary electrophoresis (CE) platform and, in the case of Qiagen’s QIAxcel Advanced System, “target all customers that are doing more than 12 samples/day,” says product manager Sebastian Bühren. The QIAxcel can handle up to 96 samples without manual intervention by iterative use of a 12-capillary array and autosampler. Other automated CE instruments, such as Advanced Analytical Technologies’ Fragment Analyzer™, can be purchased with either a 12- or 96-capillary array. Such systems often can sample from microtubes or 96-well plates, and many have built-in autosampling capabilities or are designed to be robotic-compatible.
Still others, like PerkinElmer’s LabChip® systems, for example, use an automated capillary “sipper” to load samples from up to 384-well plates onto a microfluidic chip.
Automate the slab
For Life Technologies’ E-Gel® system, “the automated part of it is the entire upstream process,” explains senior product manager Mazen Karaman. The device can be put onto an automated platform, and because the 48- or 96-well slab E-gels are dry and encased in plastic, they can be manipulated by a robotic gripper. Because the wells are very well defined, Karaman adds, it’s easy to map them on an automation platform for automatic loading.
Although larger sample sizes generally are required on an E-Gel than for microfluidic- or CE-based systems, this can actually be an asset. Purified sample can be recovered by allowing the band to run into a second cut-out well. Because the E-gel is dry when the sample gets to in the second well it can be pipetted up in water, TE, or other buffer.
There are also smaller, 12-lane E-Gels that can be used for sample recovery. These gels can be automatically imaged while running by using a companion light box and camera setups. (Lonza’s FlashGel is a similar system.) Researchers do not tend to automate these smaller systems with robotics, says Karaman.
The capital costs of E-gel systems are comparable to those of traditional slab-gel rigs, with consumables costing about the same as pre-cast gels. Microfluidic and CE systems, on the other hand, require a capital outlay of at least $10,000 plus consumables. (Consumables for the QIAxcel, which Bühren claims is “the cheapest in the field,” start at 30 cents to $1 per sample, depending on the application.)
When calculating the price of an automated electrophoresis system, ask: What price time? What price accuracy? What price reproducibility? And then decide whether it’s worth the cost.
Image: Agilent Technologies