by Caitlin Smith
Many researchers don’t give the plastic labware they use a second thought. But not all plasticware is created equal. How do you go about selecting the right tube or microplate for your experiments? And how can you be sure that these products are merely containing your samples and not (perish the thought) reacting with or contaminating them? Here are some tips on how to answer these questions for your own research.
Contaminants and leachables? No thank you.
Whether you are in the market for tubes (typically used more often for short-term reactions or storage), plates (typically used more for screening) or both, the integrity of your samples and reactions is paramount. For this reason, your choice of plasticware requires due consideration.
“One thing I think the customer needs to pay attention to is whether . . . tubes or plates were manufactured in a certified clean environment,” says David Du, product manager at Bio-Rad Laboratories. “Some are not, which means that the tubes or plates could be contaminated during the manufacturing process, which would compromise the reaction. Contaminants could include the operator’s DNA from skin or hair that could fall into a well, [as well as] PCR inhibitors, DNAse or RNAse." The manufacturing industry uses different classes of cleanrooms. “One commonly used, acceptable one is the Class 100,000 cleanroom, meaning there are less than 100,000 particles in the air per cubic foot,” explains Du. “One step further is the Class 10,000 cleanroom, which is 10 times cleaner. This is where we manufacture our high-end plates. A good manufacturer also tests plates to make sure they are free of contamination.”
Although cleanroom manufacturing is an important consideration, it doesn't guarantee contaminant-free plastic. This is because contaminants may leach out of the plastic itself. For example, the lab of Andrew Holt showed in a Science paper that contaminating agents such as oleamide (a “slip” agent that lubricates the plastic where it contacts the manufacturing equipment, to aid in release from the molding tool) and biocides (agents that inhibit growth of bacteria) can leach from plasticware into common solutions such as standard aqueous buffer, dimethyl sulfoxide (DMSO) and methanol. Furthermore, Holt and his team showed that these agents "have profound effects on proteins and thus on results from bioassays of protein function." [1] (An interview with Andrew Holt can be found on Eppendorf's website.)
Matthew Lieber, product manager for centrifugation and sample prep at Eppendorf, points out that such leachables are of significant concern because their effects are not yet well understood.
In addition, while many standard contaminants such as DNA, DNAse and RNAse are tested for routinely by both manufacturers and researchers, leachables are not. As a result, leachables can unknowingly interfere with reactions and results.
"For a very long time now, [Eppendorf has operated] without the use of any slip agents, biocides or plasticizers in the manufacturing of our tubes, tips and plates," says Lieber. Eppendorf does this by using highly polished molding equipment (to avoid the use of slip agents) and clean manufacturing rooms (to avoid the use of biocides). As for surfactants, he says, “We only use them for making colored tubes, because it’s necessary for the dye to bind to the tubes.” Lieber recommends the use of clear tubes to avoid surfactants—amber- or black-colored tubes are only functionally required to block natural light. Eppendorf also certifies the trace metals in each batch of plasticware and makes the information available to customers.
Don't lose your sample to the plastic: Low-retention surfaces
Sample vessels vary in the degree to which the inside surface of the tube or well retains sample. Thus, manufacturers strive to minimize biomolecules adhering to the vessel walls. Lieber says the new Eppendorf LoBind® product line uses a new technology to prevent this in standard polypropylene tubes. “We do ours in a different way,” he says. “Most of the low-retention tubes (and plates) on the market are coated, usually with silicone, which is a nonbinding surface. But if you apply vigorous heat or shaking to the tube, the silicone coating can actually come off. This doesn’t necessarily inhibit the reaction, but it interferes. Our LoBind tubes are . . . [made from] a two-component polymer mix, so it’s actually a modification of the polypropylene resin itself—it’s not coated at all.” Eppendorf offers two forms of LoBind tubes: one designed to work especially well for proteins and another that’s better for nucleic acids. Lieber says the difference between the two models is related to the more hydrophobic cores in proteins; LoBind tubes for proteins are designed to repel this core, resulting in less sample retention.
Storage considerations
Having tackled the weighty concerns of contaminants, leachables and sample retention, there are other, more basic, factors to consider. At ThermoFisher Scientific, product market director Ray Mercier suggests that you consider several questions. “What material and volume are you storing? How long will it be stored? How often will it be retrieved?” Mercier asks. These questions lead to issues of storage capacity in the lab, as well. ThermoFisher Scientific emphasizes maximizing your tube storage capacity. “When they are going to store a lot of samples, a lot of people don’t consider freezer space and the volume that they will store in a tube,” says Mercier. “Most customers will buy, for instance, a 2 ml cryotube because that’s what they’ve always used, but we’ve found out that over 60% of the customers that purchase a 2 ml cryotube put less than 1 ml in that tube.” ThermoFisher Scientific’s new line of storage, in keeping with its Dense Storage Initiative, lets you store more than twice as many samples in a freezer, saving space and electricity. When storage considerations include retrieval at a much later date, possibly by someone else, Mercier says “users are urged to consider prelabeled tubes and barcode labeling. If it’s important enough to store, then label it properly, so you or someone else can identify it later.” ThermoFisher Scientific also offers ID Scribe Labware Identifier, a tool that prints neatly on tubes with legible writing and unique 2D data matrix codes. Both of these systems may make life easier when it comes time to retrieve samples.
According to Mercier, “most storage tubes are made from polypropylene, which works well for samples such as blood, plasma, DNA, [samples containing] DMSO and most liquids used in research or sample collection. By contrast, plates are usually made from polystyrene for assays that are used in absorbance, fluorescence or luminescence protocols.” However, if researchers are using organic compounds such as DMSO in the plates for a long enough time for impurities possibly to leach from the polystyrene resin, he recommends that they change to polypropylene. If organic compounds continue to be a concern, Thermo Fisher Scientific also sells glass 96-tube arrays and plates.
PCR: Minimize warping, maximize heat transfer
If your research requires PCR tubes and plates, Bio-Rad offers its new Hard Shell® plates in 96- and 384-well configurations. These plates are designed for increased strength while maintaining a high-quality reaction vessel. “Generic PCR plates are made of polypropylene, which is ideal for PCR reactions but not ideal for automation, because after thermal cycling they tend to warp,” says Du. “The warped plates present a challenge to robotic handling, especially with automated pipetting, where the needle tends to crash into the plate walls.” To reconcile PCR requirements with automated pipetting, the company constructed plates from two different materials. “For the plate's reaction wells, we still use polypropylene,” says Du. “For the skirt and the shell of the plate, we use more rigid material, such as polycarbonate. This gives the plates a more rigid structure, so they do not warp with thermal cycling [or heat sealing].” These more automation-friendly plates have been used by major high-throughput labs and sequencing centers. Until recently, the Hard Shell plates only fit Bio-Rad instruments; however, Bio-Rad has started making different plate configurations that fit all major instruments. Plates also are offered with white-colored wells, which can improve the detection of fluorescent signals.
Size and shape are fundamental aspects of tubes, as are the well portions of plates. The thickness of well walls is directly related to their ability to transfer heat from the thermocycler block to the tube or well contents during PCR reactions. “These are thin-walled tubes, usually around 0.25 mm, to ensure fast heat transfer during thermocycling reactions,” says Du. “To make them thin-walled is very challenging, because you have to be very precise in the manufacturing process. We always try to make the walls thinner for faster heat transfer, but you have to balance that with reliability. Lower-end manufacturers tend to make the tube walls thicker than 0.25 mm because it is easier to manufacture, takes less expertise and costs less. But thicker walls lead to compromised PCR reactions.”
To cap, or not to cap?
Deciding whether or not to cap—and how to cap—depends on your particular experiments. Capping may be an issue during boiling, for example, when internal pressure can cause unsecured caps to open accidentally. Some manufacturers offer caps or lids designed to lock, or at least stay on under high-heat condition; these products include Eppendorf’s hinged SafeLock® tubes. Thermo Fisher Scientific’s new Cap-It-All® tool for storage tubes comes in two varieties: a handheld eight-tube model and an automated benchtop model in a 96-tube format.
Blood collection tubes
A unique solution to the quandary of whether to use lids or caps is to use a closed system. BD Diagnostics-Preanalytical Systems offers new proteomics blood-collection tubes that include cocktails of protease inhibitors to stabilize blood proteins upon collection. “There’s a huge difference between the standard EDTA blood-collection tube and these, which have very complex chemistries to stabilize proteins,” says the company’s proteomics R&D manager, David Craft. “Our P700 tube has an inhibitor to stabilize GLP1, which is used in diabetes research, especially in drug development. Our P800 tube is targeted to stabilize GLP1 as well as GIP, glucagon and grehlin.” The company’s new blood-collection tube, the P100, includes a newly designed mechanical separator in addition to the protease cocktail. “Some blood-collection tubes don’t have any separators, some have a gel, but this one has a mechanical separator,” says Craft. “It has high-density and low-density pieces that pull in opposite directions when you centrifuge it. The separator is designed with a particular density, so it will sit between the plasma and the cells. When you stop centrifuging, the high-density and low-density pieces go back to their original positions, making an O-ring that expands out and forms a seal between the plasma and the cells.”
The combination of the mechanical separator and the protease inhibitors results in a sterile, clean, stable plasma sample. “A lot of researchers are uncapping and adding their own protease inhibitors, or capping and uncapping and aliquoting their samples,” says Craft. “With P100 tubes, you don’t have to uncap to add protease inhibitors because they’re already in there at the point of collection. This is important because even in the time it takes to centrifuge a sample, degradation can occur. We are always emphasizing that stability can be an issue for some biomarkers, for example.”
Tubes for new purposes: cell culturing and electroelution
Are you interested in electroelution or cell culturing in tubes? Tubes are being used for more than PCR reactions these days—you might consider using them for other experiment types. MIDSCI (Midwest Scientific) offers the new TPP TubeSpin Bioreactors in 50 and 600 ml volumes. “These tubes are ideal for culturing suspension cell lines, as they increase the gas exchange within the tubes while the cells are in a shaking incubator,” says Graziella Mendonsa, PhD, product manager at MIDSCI. “Alternatively, they also offer the scientist options to control the amount of air flow into the cells to modulate conditions of optimal or stress environments for experimentation.” MIDSCI recently introduced FlexTubes, for more convenient protein dialysis and electroelution of nucleic acids or proteins from a gel—inside a tube. “For protein dialysis, no longer will [scientists] have to worry about piercing their expensive dialysis cassettes, or their dialysis tubing uncapping during the buffer exchange process,” says Mendonsa. “Likewise, the electroelution option with the FlexTubes permits a quick and environmentally-friendly option of nucleic acid and protein isolation for sequencing and avoids using the time-consuming and toxic reagent kit route.”
Most importantly, finding the right tubes or plates supports the investment you’ve made in your samples. “Consider the significant investment pharma companies have made in biomarkers,” says Susan Stansfield, marketing manager for clinical research at BD Diagnostics-Preanalytical Systems. “Our tubes can standardize and stabilize those biomarkers, making the drug discovery process more efficient and protecting the valuable investment that they’re making in those particular biomarkers. It’s like insurance for your biomarkers.”
Reference:
[1] McDonald, GR, et al., “Bioactive contaminants leach from disposable laboratory plasticware,” Science, 322(5903):917, 2008.
The image at the top of the page is from Eppendorf's twin.tec PCR plates.