Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
Messenger RNAs were originally thought to have the leading role when it came to translation and regulating expressing in cells. Non-coding RNAs (ncRNAs)—including micro RNAs (miRNAs) and long non-coding RNAs (lncRNAs) have been sharing the spotlight and garnering the attention of scientists. Non-coding RNAs have been identified as potential biomarkers linked to diseases such as cancer, autism, Alzheimer’s disease and others. It is the vision of researchers and clinicians to be able to detect ncRNAs in noninvasive liquid biopsies, such as blood samples (rather than more invasive tissue biopsies), for use in developing tests to better diagnose and treat diseases. Here’s a look at some advances in ncRNA tools and their applications in today’s research.
Isolating ncRNAs
Researchers commonly study ncRNAs by purifying total RNA—coding and non-coding alike—and then pulling out the ncRNA of interest using primer design and amplification. To assist in this first step of the process, RNA preparation kits are available from many tool providers. For example, QIAGEN offers a variety of kits that differ based on the sizes of RNA species isolated—the RNeasy kit isolates total RNA of 100 nucleotides (nt) and larger, and the miRNeasy kit isolates total RNA of 18 nt and larger. Other vendors offering total RNA kits include Bio-Rad Laboratories, Clontech, Epicentre (an Illumina company), MilliporeSigma, Norgen Biotek, Promega, Thermo Fisher Scientific and Zymo Research.
Amid greater interest in studying miRNAs as biomarkers isolated from biofluids, QIAGEN offers solutions for liquid-biopsy samples types. A subgroup of the miRNeasy kit is exoRNeasy, “which is focused on the purification and enrichment of exosomes and then doing the total RNA isolation from there,” says Brian Dugan, global product manager for miRNA at QIAGEN. The company also offers an miRNeasy serum/plasma kit that can be adapted for any biofluid. “We developed solutions to assay miRNAs in blood samples and other biofluids, because a lot of researchers are specifically looking at that,” says Dugan. In addition, QIAGEN recently launched a Targeted RNA Sequencing Kit for sequencing lncRNAs (and mRNAs).
Isolating ncRNA is of special interest for clinical oncology researchers, who are trying “to match treatments with the patient via biomarkers, to better personalize medicine,” says Jeffrey Franz, general manager for sample preparation at Thermo Fisher Scientific. “In addition to testing for cancer-associated mutations, for example, researchers are increasingly looking to examine RNA for information that might guide a second line of drug treatment.” But researchers looking for new treatments are also asking for higher-throughput RNA tools.
New ncRNA tools
Many total RNA preparation kits leverage the convenience of spin columns; however, sample throughput may be a limiting factor for those focused on processing numerous samples. New isolation tools, such as Thermo Fisher’s MagMAX™ mirVana™ Total RNA Isolation Kit, can help expedite the time to results by using magnetic beads. The assay can be performed manually, or it can be run in an automated workflow in microwell plates for higher throughput—up to 96 samples at a time.
Rare cell types from the immune system are another target researchers are interested in studying.
Brian Brown, associate professor at the Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, participates in the Immunological Genome Project (Immgen), which is funded by the National Institutes of Health. “One of the goals of Immgen is to determine the microRNA regulatory networks of the entire immune system and make this a public resource to the scientific community,” says Brown. “Our profiling includes many rare immune-cell subsets, such as NKT cells and gamma delta T cells, which can be so rare that we can isolate less than 10,000 cells.”
Brown uses QIAGEN’s miScript platform for miRNA profiling, because the miScript preAmp system is particularly advantageous for working with low-abundance samples. He also uses miScript PCR arrays to profile hundreds of miRNAs. Overall, Brown finds QIAGEN tools valuable because they have been very quantitative and consistent. “The miScript platform is integral to our efforts, because it enables us to perform whole-genome miRNA profiling even starting from [fewer than 10,000] cells,” he says.
Other ncRNA tools include MilliporeSigma’s RNA immunoprecipitation (RIP) assays, which isolate ncRNAs along with their binding partners, such as miRNAs and their mRNA targets. Magna Nuclear RIP™ kit from MilliporeSigma assists researchers analyzing RNAs such as lncRNA or miRNAs that associate with chromatin. And the Magna ChIRP® kits for chromatin isolation by RNA purification (ChIRP) can isolate lcnRNAs for studying their regulatory roles in disease.
Christian Nievera, senior product manager for molecular platforms at MilliporeSigma, says researchers currently are using the company’s tools to study links between ncRNAs and disease regulation. “One example for miRNAs is let-7, which plays a key role in regulating DICER and c-MYC gene expression and is a potential therapeutic target for cancer and cardiovascular diseases,” says Nievera. “Also, ChIRP analysis has been used to study long non-coding RNAs and [their] effects on gene regulation, including how the lncRNA THRIL regulates TNFα expression.”
Easier sample preparation
Sample preparation for some RNA assays has become much easier to perform. For example, Thermo Fisher Scientific’s Cells-to-CT assay performs cell lysis, targeted amplification and reverse transcription, all within the same sample well of a microplate. As such, it is geared toward pharmaceutical companies and research labs that are doing high-throughput screening. Users don’t necessarily need to isolate the ncRNA first, so the assay moves forward directly into amplification procedures, says Franz.
Abcam’s Firefly Multiplex miRNA assay platform also detects miRNAs from blood, plasma and serum samples without prior RNA isolation or purification. “We can quantify miRNAs directly from biofluids, because we’re using a bio-inert substrate that doesn’t get bound up by different proteins or lipids that are present in biofluids,” says Dan Pregibon, general manager for platform innovation at Abcam. The system detects miRNAs using gel rods, each of which has a specific barcode, as well as binding zones for the biomarker of interest. Multiple rods are mixed together with the sample for multiplex detection and then read on a flow cytometer. Assays are run in 96-well microplates, and they can detect up to 68 miRNAs in each sample, making the Firefly platform promising for high-throughput screening. Researchers can develop custom panels of up to 68 miRNAs, but Abcam also offers focus panels in specific fields, such as oncology, immunology and toxicology.
Pregibon and others at Abcam work with researchers to develop biomarker signatures using the Firefly technology. These complex, early biomarker signatures, typically comprised of approximately 10 to 50 miRNAs, can be used to screen samples for hallmarks of disease. Jordan Plieskatt, a senior research associate in the lab of Jeffrey Bethony at The George Washington University, is using the Firefly platform to develop a miRNA biomarker signature for cholangiocarcinoma (CCA), a bile duct cancer that often goes undetected until the disease is too advanced to treat successfully. He hopes a CCA biomarker signature for diagnostic or prognostic use might give patients and their doctors more time for therapeutic interventions.
Among the risk factors for CCA is previous damage to the liver—for example, from viral hepatitis or, in Plieskatt’s project, induced by parasitic liver fluke Opisthorchis viverrini, found in southeastern Asia. Plieskatt studies a population of CCA patients in rural Thailand, where this liver fluke is prevalent. He says the Firefly platform fits his needs, because the assay uses relatively small volumes (30 to 50 microliters) of biological fluids, such as sera or plasma. This helps conserve his precious supply of stored samples collected from patient cohorts in Thailand (although the assay can analyze a sample as small as 10 to 20 microliters). He also wanted an assay with no sample preparation, to increase throughput and decrease user variability. And ideally, he wanted an assay he could perform on-site in rural Thailand using a wide variety of accessible biofluids in a single workflow.
The Abcam team also worked with Plieskatt to apply the Firefly technology to his desired sample-collection system of fiber cards, onto which he spots serum and plasma samples (like the cards used for newborn blood-screening tests). To run assays, he punches 7-mm discs out of the samples he wants to analyze, and these integrate into the Firefly workflow seamlessly, replacing a liquid matrix with a dried blood spot. “Now I have a way to collect samples in the field,” he says. “I also eliminated the cold chain, because the miRNAs are stable on these fiber cards at different temperatures.” In the future, instead of taking samples home to Massachusetts for analysis, he hopes to be able to analyze locally, using a cytometer at a local Thai hospital. Beyond dried blood spots, the Firefly platform has been used to profile miRNAs in other challenging sample types, including heparin plasma and formalin-fixed paraffin-embedded (FFPE) tissue.
The only aspect of Firefly technology Plieskatt might change is adding “colorimetric tags on the hydrogel particles for a few key miRNAs, so we could read results immediately,” he says. “That way we could do preliminary tests in the field and follow up with more in-depth analyses only if needed.”
Abcam’s Firefly also can combine the power of miRNA and protein biomarkers using parallel biomarker signature panels with miRNA assays and new immunoassays, both of which use Firefly particle technology. Pregibon is excited about the prospect of researchers detecting a combination of miRNAs and proteins as biomarker signatures. “We’re talking to customers who would love to use both types of assays to monitor cancer immunotherapies, to measure both the miRNAs and the cytokines implicated in the immune response,” he says.
Look for increasing advances in our understanding of diseases and their regulation—such as how the immune system and cancer intertwine—as scientists use today’s RNA tools and apply the power of combined ‘omics to uncover the intricate regulatory webs of ncRNAs.
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