RNA Sample Prep

RNA is a notoriously unstable molecule—far more so than DNA. Nearly everyone has heard at least one horror story about working with the finicky nucleic acid—perhaps having to do with a vanishing pellet or a low-molecular weight smear on a gel. Fortunately, as the field has matured and become more mainstream, so have many of its tools and protocols. Here we look at some ways RNA researchers manage to have happy endings.

The evil four

Alkaline conditions, metal ions and heat can all promote hydrolysis of RNA molecules. Thus, RNA work typically is carried out in neutral or slightly acidic buffer, at 0°C to 4°C, and often in the presence of chelating agents such as 0.1 mM EDTA, if possible. For crucial applications—especially those that may not tolerate EDTA—an ion-exchange resin can be used to rid it of cations.

RNAses, though, are often the bane of RNA research. These ubiquitous enzymes come from the biological source from which the sample is taken—sequestered in different cellular compartments and released during lysis—and also may be introduced by handling in the lab. “They’re everywhere—on skin cells, on your hands. If you’re wearing gloves, and you touch something like your face or arm, you likely picked up some RNAses,” cautions Eric Vincent, global product manager, genomics, at Promega.

When harvesting tissue or cells, “the trick is to get the RNA out as quickly as possible or to get it into a state where the RNAses are inactive,” says Bret Robb, head of RNA research at New England BioLabs (NEB).

If it’s not possible to extract the RNA immediately, samples should be stabilized by snap-freezing—or stored in a specialized solution for preserving RNA such as AllProtect Tissue Reagent or RNAlater or in a specialized blood-draw tube such as PreAnalytiX’ PAXgene Blood RNA Tube to “ensure not only that the RNA is safe from degradation, but also that … the cells do not start expressing any transcripts due to change in temperature or buffer conditions,” says Martin Schlumpberger, associate director of scientific applications, RNA product development, at Qiagen. PreAnalytiX is a joint venture of Becton, Dickenson (BD) and Qiagen.

Lysis buffers in most commercial RNA-isolation kits, such as Qiagen’s RNAEasy, are “designed to inactivate any RNAses that come with the sample,” Schlumpberger explains. After everything is homogenized and in the buffer, “it’s pretty safe.” Similarly, the chemicals used in DIY protocols, such as guanidine thiocyanate, phenol/chloroform and SDS, are protective of RNA.

It’s after the RNA is purified away from chaotropic chemicals that the solution becomes most vulnerable to environmental RNAses. That is when special precautions must be taken.

Good lab practices

The key to successful RNA work is common-sense mixture of techniques used for qPCR and for sterile mammalian culture, minus the laminar flow hood. Wear disposable gloves. Keep RNA work separate, especially from things like plasmid preps and bacterial work. And in general try to minimize contact with things that you aren’t 100% confident are clean.

How to achieve such confidence? NEB’s RNA labs generally use dedicated disposables. They filter solutions into certified RNAse-free bottles (rather than autoclaving, because an autoclave can release RNAses from microbes in the solution, and RNAses have been known to renature after autoclaving). They use filtered pipettes and disposable plastic test tubes for just about everything. It’s easier now, Robb says, because there are a lot more kits, reagents and plasticware available that have been produced and tested with RNA work in mind than there were 10 to 15 years ago.

“There used to be a lot of apprehension about making solutions—you didn’t want to breathe when you were weighing things out and stirring things for fear of contaminating them and getting RNAse in there,” he explains. Now, you can purchase everything from water to 1M Tris and 5M NaCl that have been tested for RNAse contamination. “I think it adds another level of security for doing RNA work.”

Many nuclease-free plastics have been tested and certified by third parties such as Mo Bio Laboratories. “For established manufacturers we used to reject a lot more products a decade ago,” says Mo Bio’s services lab manager Carl Tsang, “We still get the smaller manufacturers who lack the resources, capabilities, or expertise to establish a consistent contaminant/nuclease free production.” The company has worked to educate manufacturers about how to identify and eliminate sources of contamination.

For labs that use glassware, it’s recommended that these be baked (or dry-autoclaved at high heat) at a minimum of 180°C for several hours to permanently destroy RNAses.

There are a number of commercial kits, such as NEB’s RNAse Contamination Assay Kit, which enable researchers to query the presence of RNAses.

Additions

Diethyl pyrocarbonate (DEPC) has long been used to treat water and solutions to inactivate RNAses. “Then you autoclave to destroy the excess DEPC—it’s unstable at high temperatures—it releases CO₂ and then basically goes away,” says Schlumpberger. Yet many people do not trust working with the carcinogenic chemical, and Schlumpberger warns that if that’s not done properly residual DEPC will also inactivate the RNA.

Surfaces, pipettors, gel boxes, combs and other things that can’t be autoclaved can be treated with a product such as RNase Away decontamination reagent and then rinsed with RNAse-free water.

Vincent recommends routinely using RNAse inhibitors like Promega’s RNAsin, both in purified RNA and in most enzymatic reactions. RNAse inhibitors specifically bind to and inhibit the common RNAses found in a lab environment—“the heavy hitters” like RNAse A, B and C and human placental RNAse. “It’s that extra piece of insurance, because you never know that you’ve introduced an RNAse until it’s too late, and your results come back negative,” he says.

RNA work doesn’t have to be scary. Good results begin with understanding the pitfalls—like high pH, heat, metals and endonucleases—and knowing how to avoid them.