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.
Better characterization of the transcriptome brings researchers one step closer to understanding the regulation of disease states. A great deal of focus is placed on ribonucleic acids (RNAs) and their interplay with other molecules. The term RNA encompasses a wide range of molecular sizes, from larger messenger and ribosomal RNAs to micro and small nucleotide RNAs (miRNAs and snRNAs). Increasing interest in understanding the regulatory roles of noncoding small RNAs has prompted the need for better tools to extract and isolate them for research studies. Though the kits do not differentiate between types of small RNAs, the various offerings provide different levels of RNA isolation and enrichment. Here is an overview of commercially available kits for small-RNA isolation, as well as insight into recent reagent and application improvements.
Filtering through the options
Many companies offer kits for isolating total RNA, and a handful of them also offer kits specifically for small RNAs (sometimes designated as miRNA kits, even though they may also contain other types of small RNAs). The convenient spin-column format is common, but kits are also available in multiwell plate and magnetic-bead formats for higher throughputs. Like other types of nucleic acid isolation kits, there are choices of size in terms of input volume, with such designations as mini or micro. Examples include Life Technologies' MirVana miRNA Isolation Kits, QIAGEN’s miRNeasy kits, Sigma-Aldrich's mirPremierTM microRNA Isolation Kit, Clontech's miRNA Purification and Isolation Kit, Roche's High Pure miRNA Isolation Kit and Norgen Biotek's microRNA Purification Kit. Some companies offer kits for particular sample types, such as cells and tissues, formalin-fixed paraffin-embedded (FFPE) tissues and serum or plasma.
The traditional approach to isolating RNA stems from procedures that work well with DNA: phenol extraction followed by isolation onto a silica filter and ethanol precipitation. Phenol removes proteins from the lysate and enables extraction of all types of nucleic acids. Although this is a trusted method, some tool providers are moving away from extraction with phenol and other organic solvents because researchers prefer not to use or dispose of hazardous chemicals. For example, Norgen Biotek, Roche, Sigma-Aldrich, Promega and Life Technologies offer kits that are free of organic solvents.
Protocols based on silica filters are often not sufficent to recover total RNA, including small RNAs, however; that is why many kits still use both phenol and silica when targeting total RNA. Consequently, those that are going phenol-free are changing their filter or their extraction chemistries—or both. For example, Norgen Biotek's matrix is made from a patented silicon carbide resin that is even better for isolating total RNA, including small RNAs. One advantage is that silicon carbide gives greater sensitivity for small RNAs. Another is that it simplifies the process: You don't need to use phenol, as it retains the small RNAs much better than silica does. "Silicon carbide binds RNA irrespective of size and at an equal rate," says Nezar Rghei, vice president of business development at Norgen Biotek. In addition, when the RNA is small, it binds RNAs with no bias as to their GC content, so there is no bias of recovered RNAs. It has been shown that for small RNAs, the combination of phenol and precipitation-based isolation can show selective loss of low-GC content miRNA, whereas no significant bias has been observed for methods utilizing phenol and silica [1]. QIAGEN also offers phenol-free extraction of miRNA using silica in a variety of kits including the AllPrep DNA/RNA/miRNA Universal Kit.
Promega's new Maxwell® RSC miRNA Tissue Kit is an automated RNA-extraction system that provides a look at the full range of RNA in a sample, not only small RNAs. "It provides good coverage of the miRNA in the sample, which then lets the user normalize small RNA to the larger messenger RNA," says Chris Moreland, global product manager in integrated solutions at Promega. Besides providing higher throughput, the system is especially useful for researchers looking at up- or down-regulation of small RNAs, as it gives them a representation of all RNA species. "Because they may not be targeting a specific set of miRNAs, they just want to know who's at the party, essentially, and this allows us to do that," says Moreland.
Zeroing in on miRNAs
As researchers continue to explore diverse sources to uncover regulatory small RNAs, tool providers are modifying and improving their kits and methods to assist scientists. Exosomes, plasmosomes and other types of extracellular vesicles are particularly rich in miRNAs. "When you lyse vesicles from plasma or serum, it's predominantly small RNA that you find, especially miRNA," says Rghei.
QIAGEN's new exoRNeasy product line is designed to "extract pure exosomal RNA, including miRNA, without any nonvesicular miRNAs that are present in body fluids like blood and serum," says Martin Schlumpberger, QIAGEN's associate director of scientific applications. The patent-pending technology captures exosomes and other extracellular vesicles by binding vesicles to a membrane. This step helps to solve two challenges inherent in isolating miRNAs from exosomes. It removes RNases and potential inhibitors of RT-PCR and other other applications that can be present at high levels in exosome-containing serum and plasma; and it also removes larger protein complexes that typically contaminate vesicles enriched by precipitation-based methods. The membrane-bound vesicles are then lysed to release their RNA content.
Cells and tissues are not the only starting material for small-RNA extraction. Researchers are exploring liquid biopsy using plasma, urine or cerebrospinal fluid as possible sources, according to Doug Horejsh, senior scientist in research and development at Promega. "Particular miRNAs have been shown to be up- or down-regulated in the plasma of cancer patients," he says. "I think that as the biomarker discovery world [opens] up, people are going to look much more closely at liquid biopsies."
Important considerations
The first considerations for choosing a small-RNA prep kit are sample types and input volume. "What small-RNA fraction are people looking for—from body fluids, tissues or exosomes?" asks Schlumpberger. "All of these different starting materials require different approaches or kits." Rghei thinks that it's important to also consider sensitivity of the reagents to isolate samples. For example, Norgen Biotek's silicon carbide columns bind RNA with picogram sensitivity and without losing linearity, such that you can recover the small RNA from as little as a single cell.
Also consider whether you want to study small RNAs in isolation or in the context of a range of RNA species. "[Promega's system spans] all RNA, so you're getting the complexity across all of the different noncoding RNAs," says Horejsh. In the future, he expects to see more focus on noncoding RNA species, in addition to miRNAs and siRNAs. "As you look across some of the other RNA species, there will be opportunities for researchers that use a total RNA approach, [vs.] other, more focused approaches," he says.
Whether you're delving into liquid biopsies, harvesting miRNA from exosomes or looking for the most sensitive way to isolate small RNA from a single, cultured cell, one thing is certain. The range and technical features of the various kits available for isolating small RNAs today are varied, plentiful and continually improving. Exciting characterizations of noncoding small RNAs are just waiting to be uncovered!
Reference
[1] Kim, Y-K et al., “Short structured RNAs with low GC content are selectively lost during extraction from a small number of cells,” Mol Cell, 46:803-895, 2012. [PubMed ID: 22749402]