Amber Dance is an award-winning freelance science writer based in Southern California. She is the ALS (Lou Gehrig’s disease) reporter for the Alzheimer Research Forum. She contributes to The Scientist and Nature journals, and has written about topics ranging from record-breaking rocks to bizarre new ant species.
If you want to understand the proteins your organism of interest is making, look to its RNAs.
Analyzing RNA expression, by quantitative PCR or sequencing, has become commonplace. As Robert Farrell, Associate Professor of Biology at Penn State University in York points out, “There is no such thing as PCR for proteins.” That makes mRNAs the best way to obtain protein expression data.
The first and most crucial step is to isolate high-quality RNA, avoiding the ribonucleases that are the bane of RNA scientists everywhere. Some protocols use guanidinium-based solutions and organic compounds to extract RNA. Many commercial kits rely on glass columns that capture the nucleic acid.
If your tissue is soft and easy to homogenize, like the brain or cultured cells, you should have a relatively easy time. But other samples are trickier customers, interfering with purification protocols because they are difficult to homogenize, crammed with endogenous RNases or contain fragmented RNA. In the past several years, biotech companies have developed a variety of specialized kits for certain samples, like plants or microbes. In particular, the rise of kits to harvest RNA from formalin-fixed paraffin-embedded (FFPE) tissues has opened pathological libraries to RNA analysis.
Here are tips for making the most of these troublesome RNA-extraction scenarios:
Pancreas
Among animal tissues, the pancreas is notoriously difficult because it is chock full of RNases that may destroy the nucleic acids before you manage to access and homogenize the organ. You’ll want to get the tissue flash-frozen or into a lysis buffer, containing nuclease-neutralizing compounds like beta-mercaptoethanol or phenol, as soon as possible. Many scientists prefer Life Technologies’ RNAlater®, a nontoxic solution that stops endonucleases. It’s useful not only for the pancreas, but also for other animal tissues, plants and bacteria, and it’s particularly convenient when collecting samples in the field.
Small starting material
You may have only one or a few cells to start from. Fortunately, robust enzymes for isolation and PCR make this possible, says Farrell, author of the book “RNA Methodologies: A Laboratory Guide for Isolation and Characterization.” The key is to keep volumes small, so the tiny sample isn’t diluted. Life Technologies’ Single Cell-to-CT™ kit (CT stands for “cycle threshold,” which refers to the next PCR step) keeps all the reactions in a single tube, so nary a nanogram is lost during pipetting or washing.
It can help to amplify your sample, after converting the RNA to cDNA but before the final PCR assay. With Roche Applied Science’s RealTime ready™ cDNA Pre-Amp Master kit or Life Technologies’ TaqMan® PreAmp Master Mix Kit, plus a primer pool to match your genes of interest, you can boost your cDNA quantity.
FFPE tissues
Typically, the pathologists who prepared your suddenly accessible FFPE slides—possibly decades ago—did not have RNA in mind. “You’re dealing with samples that are messed up from the get-go,” says Jami English, a senior research scientist in research and development at Promega. Endonucleases likely fragmented the RNA before fixation, the formalin crosslinked the RNA with proteins, and the whole sample is encased in wax.
One common method to remove the paraffin wax is with smelly, toxic xylene, as with Life Technologies’ RecoverAll™ kits. The company’s MagMAX™ kit uses alternative, less unpleasant chemistry to dissolve paraffin.
There are a couple options for dealing with the fragmented RNA in FFPE samples. As with small samples, you can try a pre-amplification step. Timothy Nilsen, director of the Center for RNA Molecular Biology at Case Western Reserve School of Medicine in Cleveland, recommends size-fractionating the isolated RNA, by column or gel, to purify the largest pieces. Promega’s ReliaPrep™ FFPE kits are good at collecting these larger fragments, adds Doug Horejsh, a senior research scientist at the company. Or, you can design downstream PCR assays to amplify the smallest possible fragment, the likeliest to be present in measurable amounts, suggests Suzanne Kennedy, director of research and development at MO BIO Laboratories. For example, you might amplify the first 70 base pairs of a gene.
Plants
Isolating RNA from plants is complicated by their hard-to-break-down cell walls as well as the presence of polyphenols and other compounds that co-purify with nucleic acids but interfere with later assays, such as PCR. MO BIO and others offer kits with specialized chemistry to remove these inhibitors. Farrell recommends checking if the kit has been tested in your particular tissue type; components like bark or berries likely have different requirements. To ensure the prep was inhibitor-free, you can do PCR for a standard reference gene that you know should be expressed.
Microbes
Like plants, microbes possess a tough-to-crack cell wall. Attacking it with lysozyme could be a problem, because the bacteria are still alive at the start of the reaction and could alter their RNA expression in the time it takes to lyse them, notes Richard Fakete, senior manager of research and development for molecular biology at Life Technologies. Life Technologies’ RiboPure™-Bacteria Kit and MO BIO’s UltraClean® Microbial RNA Isolation Kit both use bead-beating—vortexing with tiny beads—to break the cells open quickly.
MicroRNAs
During the last decade, scientists have developed interest in microRNAs and other mini-nucleic acids, some a mere 20 nucleotides long, notes Stephen Roemer, a senior manager for product development at Thermo Fisher Scientific. Not all kits do a good job of isolating the tiny pieces, he says. Thermo Fisher’s SurePrep™ kits use a special silicon carbide resin to bind the RNA, even micro species. Similarly, Life Technologies’ mirVana™ kits isolate both small and large RNAs.
Overwhelmed by the options? Some scientists say that using a specialized kit ensures high-quality RNA, but others believe basic kits work just as well. Aside from plant tissues, where special kits help, “all these products are pretty much the same,” says Nilsen, co-author of “RNA: A Laboratory Manual.” To determine whether a specialized kit meets your needs and budget, Farrell recommends testing the free samples most companies provide. That way, you can zero in on exactly the right protocol for your samples.