by Mike May
 An Agencourt researcher utilizes the company's SPRI (Solid Phase Reversible Immobilization) paramagnetic bead-based technology to isolate RNA. |
These days, RNA forms the root for a large collection of abbreviations and acronyms, composed of old favorites like mRNA and new variations such as miRNA and siRNA. Even that collection doesn’t tell the whole story, because nature itself mixes RNA quite extensively. In discussing mRNA in the January issue of the Bulletin du Cancer, Philippe Jeanteur, MD, PhD, of the Institut de Génétique Moléculaire de Montpellier in France, writes: “... alternative splicing brings about an important vulnerability to mutations, which is at the origin of many pathologies, including cancer.” There are more than letters at stake: what happens with RNA could mean life or death.
Although analytics usually gets more play than extraction, finesse is demanded in simply getting out the material in RNA research. “RNA is less stable than DNA,” says Todd Lombardo, product manager at Agencourt Bioscience, a Beckman Coulter company located in Beverly, MA. In fact, RNA can turn downright tricky. “Some say: ‘If you look at it funny, it will degrade and disappear in extraction,’” says Hemanth Shenoi, PhD, strategic marketing manager at Promega in Madison, WI. For example, to isolate RNA from pancreatic tissue, a researcher has only a minute — maybe less — to flash freeze the sample or homogenize it and get it in extraction buffers. Otherwise, enzymes in the tissue start to break down the RNA, and the researcher ends up with fragments. The same thing happens when trying to isolate RNA from blood.
Beckman Coulter’s Agencourt RNAdvance chemistry uses SPRI (solid phase reversible immobilization) to capture total RNA from samples. “This can be used for tissues, blood, in vitro reactions, even formalin-fixed, paraffin-embedded samples,” says Lombardo. In this approach, beads grab the RNA and a magnet grabs the beads. Then, something as simple as water can elute the RNA off the bead. (RNAdvance is optimized to work with Beckman Coulter Biomek NX or NXP workstations.)
 Caliper scientists prepare RNA samples for analysis on the company’s LabChip 90 System. |
Researchers can also pull out RNA with Promega’s Maxwell 16, which Shenoi calls “an automated system for RNA extraction.” He adds, “It can work with 1 to 16 samples, and it runs the extraction in 30 to 45 minutes.” ?It does require a bit of hands-on work, though, because the scientist must make a lysate and run a proprietary DNA-removal step before loading the samples on the Maxwell 16. “The clearing agent for DNA eliminates several downstream steps,” explains Eric Vincent, PhD, product manager at Promega.
The Promega process also creates very clean RNA. According to Shenoi, “Our studies find fewer than 0.1 copies of genomic DNA present in 100 nanograms of purified RNA.”
Turning RNA into IVD
If scientists can capture and keep RNA as it comes from a sample, the resulting material can be used for in vitro diagnostic (IVD) testing. “In some conditions, gene-expression levels can be measured to indicate disease status or prognosis,” explains Marie McCluskey, PhD, global product manager for pre-analytical systems/IVD at QIAGEN in Hilden, Germany.
It’s already possible to use RNA as a diagnostic. For example, the PAXgene Blood RNA system from PreAnalytiX — a joint venture of BD in Franklin Lakes, NJ, and QIAGEN — is a manual kit that carries the CE label for use in Europe and FDA clearance for use in the United States. It consists of blood-collection tubes containing an RNA stabilizer from BD and a cellular RNA purification kit from QIAGEN. “The aim of PreAnalytiX,” says McCluskey, “is to provide complete systems for the collection, stabilization, transport, and storage of human specimens, and purification of nucleic acids from these samples.” According to McCluskey, specimens collected with this technology can be kept at room temperature for three days, and in vivo RNA gene-expression levels are preserved and high-quality RNA still can be extracted.
In May, PreAnalytiX will also offer an automated RNA solution for the PAXgene Blood RNA System with their CE-IVD marked kit in Europe. The product will combine the PAXgene Blood RNA System with QIAGEN’s QIAcube instrument, facilitating automation of the purification process.
Getting more from gene expression
 Loni Pickle, Invitrogen associate scientist, uses the mRNA Catcher PLUS kit post-transcription to catch messenger RNA before performing rtPCR in the company’s Carlsbad, California laboratories. |
Typically, scientists gather RNA to study gene-expression levels. Researchers can automate much of this process by combining Beckman Coulter’s SPRI technology with its GeXP Genetic Analysis System. “This 8-capillary system does gene expression on multiplex samples,” says Keith Roby, strategic marketing applications manager at Beckman Coulter (Fullerton, Calif.). “It’s great for researchers who know what they want to look at and don’t want to spend lots of money looking at the whole genome.”
Scientists also explore gene expression with real-time PCR. “That was a niche until a few years ago,” says Seth Cohen, PhD, director of application sciences at Caliper Life Sciences in Hopkinton, MA. “Today, it appears in studies from target validation and screening through clinical uses.”
Researchers can use Caliper’s RNA assays on the company’s LabChip 90 to characterize and quantify RNA for downstream, real-time PCR or microarray studies. This device uses the company’s sipper technology to automatically load samples from microplates.
Some companies take a different approach to assessing gene expression. For instance, Illumina, in San Diego, CA, offers both microarray and sequencing-based applications for gene-expression profiling. Using its sequencing technology, Illumina is developing an application called mRNA-Seq, which sequences mRNA. “For some time, researchers have used microarrays for gene-expression profiling, which relies on the manufacturer to design the best probes,” says Shawn Baker, PhD, senior product manager for gene expression at Illumina. “The state of knowledge of the transcriptome, however, is constantly changing.” According to Baker, Illumina updates the information contained on their human, mouse, and rat arrays about every two years.
Using an alternative sequencing-based approach for gene-expression profiling, mRNA-Seq simply cranks out the sequence of the transcripts. “This way,” says Baker, “you don’t require a priori sequence knowledge. mRNA-Seq captures the mRNA and sequences it all.” He adds that the sequencing portion is highly automated, taking 2.5 days to run, which is about the same amount of time seen with microarrays. “This doesn’t mean that RNA sequencing will push microarrays out of the loop,” says Baker. “Scientists will likely combine both in their experimental plans, using Illumina arrays to quickly and inexpensively identify samples of interest, and then follow up with mRNA-Seq to gather a deeper view of the transcriptome of those samples.”
For Illumina’s gene-expression microarrays, which cost as little as $100 per sample, “researchers can expand experimental designs with more replicates, more power, and better results,” explains Baker. mRNA Seq — although not yet launched — might cost between $1,000–2,000 per sample. However, scientists might be willing to pay the higher price given the amount of information they will retrieve from their samples, which Baker calls “information that is simply not available from any array on any other platform.”
Measuring more RNA
If a researcher collects the entire transcriptome, only about 2 percent of it is mRNA. That leaves lots of other RNA — including small and non-coding RNAs — to be identified.
“A scientist could use several approaches to identify small RNAs,” says Larry McReynolds, PhD, senior research scientist in the division of RNA Biology at New England Biolabs in Ipswich, MA. “They could use an RNA ligase to join RNAs for amplification or cloning.” In addition, McReynolds and his colleagues see researchers using novel RNA-binding proteins to purify double-stranded RNAs. “This can be used to identify new double-stranded RNAs or to measure selected RNAs,” McReynolds says.
These small RNAs also end up in clinical research. “Small-RNA research is more mainstream now,” says G. Brett Robb, PhD, staff scientist at New England Biolabs. “They can be used as biomarkers when collected from tumor samples and characterized.”
Other companies also want to grab those small RNAs. “Our original kits threw away any RNA that was less than 200 base pairs long,” says Chris Adams, PhD, R&D manager at Invitrogen in Carlsbad, CA. In short, RNA that didn’t code for proteins was considered unimportant. “Now, we know that the noncoding RNA does a lot,” says Adams. “If there’s a function involved in gene expression, we will probably find noncoding RNAs that play some role.”
Consequently, Invitrogen makes products that can be used to find that RNA. For example, the company makes an miRNA-profiling platform, miRNA microarrays and labeling kits, and real-time PCR products for small RNAs. “We also recently entered into a licensing agreement with IMBcom at the University of Queensland to commercialize noncoding RNAs,” says Amy Cuneo, product manager for epigenetics at Invitrogen. “This includes tens of thousands of unique human and mouse sequences of noncoding RNAs that will help scientists identify the most-relevant ones.”
Tools for collecting all RNAs face some serious challenges. First, noncoding RNAs cover a wide size range, from just a bit more than a dozen nucleotides up to 100,000. Moreover, they often lack the poly-A tail that helps kits find mRNA. In addition, post-transcriptional splicing makes even more RNA that needs to be identified. Consequently, Invitrogen hopes to eventually make tools that will pull out all kinds of RNA from one sample.
Scientists will need this wide variety of tools to find all types of RNA, where it resides, how it functions, and how it can be used.