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.
Epigenetics researchers study chemical alterations and other regulatory mechanisms that affect gene expression but do not involve changes in nucleotide sequence. Traditional epigenetic factors have included DNA methylation and chemical modifications of the histone molecules that help to maintain DNA structure. But there is another important courier of epigenetic regulation, as well: RNA, and in particular, noncoding RNA molecules (ncRNAs). Although there are many types of ncRNAs, the most commonly encountered varieties are the relatively short (about 20 to 25 nucleotides) small-interfering RNAs (siRNAs) and microRNAs (miRNAs) and long ncRNAs (more than 200 nucleotides).
Whether long, small or micro, ncRNAs by definition are not translated into proteins, yet fulfill important regulatory roles in gene expression. MicroRNAs, for instance, can dampen expression of complementary mRNA transcripts. Long ncRNAs, though less well characterized than their shorter cousins, are increasingly understood to carry out a range of important functions, too, such as the X inactivation-related Xist transcript that recruits epigenetic regulatory complexes to the genome.
Awareness of the significance of ncRNA-mediated regulation has grown with the rise of massively-parallel “next-generation” sequencing technologies (available from Illumina, Life Technologies, 454/Roche Applied Science and Pacific Biosciences) that can interrogate those molecules. “Epigenetics is a very exciting but underappreciated field—until now,” says Nitin Puri, senior product manager at Life Technologies. “In some ways, we did not know how impactful it was. But next-generation sequencing is clearly pointing out the importance of epigenetics and [its] role in regulation of protein coding regions.”
Sequencing remains a key tool for noncoding RNA researchers. But it’s not the only one. Here we review some of the primary tools for noncoding RNA research.
Measuring ncRNA expression
One approach to studying ncRNA expression is simply to sequence the cell’s entire transcriptome—a method called RNA-seq. The method lets researchers survey all (or a fraction of) the cells’ transcripts, and to compare one sample to another. But while it offers tremendous breadth, RNA-seq suffers when it comes to sequencing depth—akin, say, to studying the night sky with low-magnification binoculars.
One alternative is to zoom in with a kind of high-power molecular telescope. That’s where RNA-Capture-Seq comes in. In RNA-Capture Seq, researchers use tiling microarrays to magnify the signal of a particular region of the transcriptome, which they then resolve by deep sequencing.
The method offers an advantage over RNA-seq in its ability to detect ncRNAs that are rare or otherwise difficult or impossible to study. “I think the RNA-Capture-Seq methodology is critical for the characterization of noncoding loci, which are commonly cell-type specific or lowly expressed and therefore not amenable to conventional whole-transcriptome sequencing,” says Marcel Dinger, head of clinical genomics and genome informatics at the Garvan Institute of Medical Research in Australia.
Conventional whole-transcriptome sequencing also does not achieve adequate depth of sequencing coverage to quantify low-abundance ncRNAs or even to detect some of them. In fact, RNA-Capture-Seq has uncovered many “new” ncRNAs from regions of the genome that were previously thought to be junk” [1].
Gene expression microarrays and real-time quantitative PCR reagents for ncRNAs are also available. For example, Agilent Technologies offers microarrays for humans and mice containing probes for long ncRNAs. “A version of these arrays was used to show that long ncRNAs act in the circuitry controlling pluripotency and differentiation,” says Yong Yi, Agilent’s director of marketing for NGS and gene expression microarrays [2]. Gene expression tools are also available from Affymetrix, Exiqon, Life Technologies and Miltenyi Biotec, among others.
Assessing function
After you identify an ncRNA, you need to figure out what it does. Dinger, for instance, is working to unravel the role of ncRNAs in disease, especially how ncRNA is involved in cancer and other “complex diseases, where conventional genetics seems to only explain a small part of the picture,” he says. His team is trying to determine whether particular ncRNAs are specific to particular diseases, and how those ncRNAs function. To do that, they use functional screens.
“Due to the large numbers of ncRNA candidates typically detected by RNA sequencing, and even more in capture-based methods, high-throughput functional screening is a key experimental approach,” says Dinger.
Screens to test a particular ncRNA for functionality may involve transfecting cells with a viral vector to overexpress or knock down the ncRNA of interest. If an effect is observed within cells, researchers can pursue more targeted experiments to understand the mechanism of ncRNA function. Life Technologies, for example, offers Silencer Select siRNA tools for silencing your ncRNA of interest.
Researchers specifically interested in microRNAs can also probe function using miRNA mimics and inhibitors, which artificially up- and down-regulate a given miRNA, respectively. Such reagents are available from Life Technologies, QIAGEN, Sigma-Aldrich and Thermo Fisher Scientific, among others.
Scouting location
Recent advances in fluorescence in situ hybridization (FISH) techniques, particularly in improved specificity and sensitivity, make FISH an attractive tool for studying subcellular localization of ncRNAs, as well, says Dinger. FISH is a microscopy-based technique that uses specific antibodies or nucleic acid probes that are tagged with fluorescent molecules to locate a molecule of interest in tissue samples with a fluorescence microscope. The Stellaris® RNA FISH Probes from Biosearch Technologies, for instance, let researchers visualize individual RNA molecules. The probes consist of a set of fluorescently labeled oligonucleotides of different sequences that bind along target transcripts, allowing detection, localization and even quantification of the labeled RNAs in single cells.
Whatever technique they use, epigenetics researchers can be confident in this, at least: Their chosen field has legs. “Work in this area is only beginning,” says Yi. “There is still much to learn about epigenetic modifications and the role ... ncRNAs play in gene regulation.”
References
[1] Mercer, TR, Gerhardt, DJ, Dinger, ME, Crawford, J, Trapnell, C, Jeddeloh, JA, Mattick, JS, Rinn, JL, “Targeted RNA sequencing reveals the deep complexity of the human transcriptome,” Nat Biotechnol, 30(1):99-104, 2011. [PubMed]
[2] Guttman, M, Donaghey, J, Carey, BW, Garber, M, Grenier, JK, Munson, G, Young, G, Lucas, AB, Ach, R, Bruhn, L, Yang, X, Amit, I, Meissner, A, Regev, A, Rinn, JL, Root, DE, Lander, ES, “lincRNAs act in the circuitry controlling pluripotency and differentiation,” Nature, 477(7364):295-300, 2011. [PubMed]