Jeffrey Perkel has been a scientific writer and editor since 2000. He holds a PhD in Cell and Molecular Biology from the University of Pennsylvania, and did postdoctoral work at the University of Pennsylvania and at Harvard Medical School.
In the classic model of oncogenesis, cancers arise through the slow build-up of mutations, freeing cells from the usual rules of behavior and allowing them to grow unchecked. Many such mutations impact key regulatory proteins—tumor suppressors, proto-oncogenes and the like. But they don’t have to; mutation of noncoding RNA genes can be equally damaging—in particular, the class of noncoding RNAs called microRNAs.
MicroRNAs (miRNAs) are short, 22-nucleotide regulatory transcripts that bind to messenger RNA targets and either inhibit their translation into protein or induce their degradation.
As such, they can influence gene expression just as surely as transcription factors can, including that of key regulators.
According to a 2014 review by Sean Lawler at Brigham and Women’s Hospital in Boston, the first clue linking miRNAs to cancer was published in 2002, with the discovery that miR-15 and miR-16 were deleted in chronic lymphocytic leukemia, leading to inappropriate upregulation of their nominal target, BCL2 [1]. “Since then it has been documented that microRNAs have roles in all of the cancer hallmarks defined by Hanahan and Weinberg, and are implicated in the clinical management of cancers at every stage,” Lawler writes.
Today, researchers have identified thousands of microRNAs across hundreds of species—the current miRBase database (version 21) includes more than 28,000 entries—and they naturally are keen to study their biology, as well as their utility as biomarkers of disease, prognosis and therapeutic response. A diverse array of molecular tools is now commercially available to assist researchers in better understanding the roles of miRNAs.
Mimics and inhibitors
Most miRNA tools fall into two basic categories, miRNA manipulation and miRNA expression analysis.
In the manipulation category are miRNA mimics, inhibitors and expression constructs. Available from Thermo Fisher Scientific and Dharmacon, GE Healthcare’s Life Sciences business, among others, mimics and inhibitors are synthetic molecules that, as their names imply, either mimic the expression of a microRNA (for example, to measure the impact of a missing miRNA) or block its ability to regulate target genes. Expression constructs enable researchers to exert longer-term influence on target cells through the transcription of one or more miRNA genes.
Dharmacon, for instance, offers both mimics and inhibitors under its miRIDIAN brand; these can be delivered to cells via simple transfection. According to Melissa Kelley, research and development manager at Dharmacon, researchers could, for instance, use both mimics and inhibitors in separate wells under the same conditions, looking for opposing phenotypes to give greater confidence in the results.
Dharmacon also offers lentiviral-expressed mimics called shMIMIC lentiviral microRNAs, for use in cells that cannot easily be transfected or in experiments requiring long-term miRNA activity. According to Kelley, shMIMIC lentiviral microRNAs are short hairpins that are expressed and processed in the cell to mature miRNAs, which can be used to assess the impact of exogenous miRNA expression in cells—to verify that expressing a specific miRNA restores normal growth, for instance.
shMIMIC lentiviral microRNAs are available with inducible or constitutive promoters as individual reagents and in pooled libraries. In the latter case, cells are transfected with a pool of shMIMIC vectors—2,555 in the case of the human pool—and grown in the presence and absence of some treatment, such as a candidate therapeutic. The cells are then harvested and sequenced to identify those miRNAs that were either enriched or depleted in the population as a result of the treatment, compared with the control.
“Instead of an array where every well has one reagent or one mimic, with a pool essentially each cell is a ‘well,’” Kelley explains.
Expression tools
As with all RNAs, researchers can quantify miRNAs using next-gen sequencing (NGS), microarrays, quantitative PCR (qPCR) and in situ hybridization (ISH), among other methods.
NGS and microarrays cast the widest net for miRNA measuring expression changes, and they typically are used in discovery research—for instance, to identify transcriptional signatures of resistance to a therapeutic. Both provide genome-scale coverage, but NGS is “hypothesis-free,” says Sara Brown, TaqMan product manager at Thermo Fisher Scientific. “You can identify new miRNAs you didn’t know about. The downsides are the workflow and the price and the bioinformatics.”
qPCR offers the benefits of low cost, sensitivity and throughput, and it is a popular choice for validating candidate miRNA signatures in larger sample sets, whereas ISH enables researchers to quantify transcripts in their morphological context, says Guy Afseth, director of oncology product marketing at Affymetrix (now part of Thermo Fisher Scientific).
Affymetrix offers solutions at both end of this spectrum, including both GeneChip® microarrays in one- (cartridge), four- (strip) and 96- (microtiter plate) sample formats, as well as ISH assays capable of fluorescently quantifying one-miRNA and two-mRNA targets simultaneously (the ViewRNA miRNA ISH Cell Assay).
ViewRNA assays are based on a branched-DNA hybridization scheme that amplifies signal intensity up to 8,000 times, and according to Afseth, the protocol is largely the same for miRNA as for other transcripts. But because miRNAs are so short, there’s no flexibility in probe placement, meaning sensitivity and specificity can suffer. “The miRNA probably has to have decent expression before you can start to detect it.”
Johanne McGregor, director of strategic marketing for the expression business unit at Affymetrix (now a part of Thermo Fisher Scientific), says the company’s current miRNA microarray design (version 4.0, based on miRBase 20) includes not only probes for every transcript in the miRBase database but also other short noncoding RNAs, such as snoRNAs and scaRNAs, “to allow researchers to get a wider view of the noncoding RNA world.” Bespoke designs also are available through the company’s MyGeneChip™ custom microarray program, McGregor adds.
Thermo Fisher Scientific offers two types of qPCR assays for miRNA detection, Brown says. First-generation TaqMan assays used a miRNA-specific stem-loop primer to create a cDNA copy of the miRNA transcript. That offers an advantage in terms of specificity, Brown says, but cDNA synthesized to quantify one miRNA could not be reanalyzed to search for another—a potential problem when dealing with precious clinical samples. “If you had 50 miRNAs [you wanted to test], you would need 50 times the amount of sample.”
More recently, the company launched a second-generation strategy based on universal primers, which amplify all miRNAs in a sample, even those that had not been discovered when the cDNA was prepared. “You can basically store the cDNA, and it can be used in the future on any miRNA you want to study” shares Brown.
According to Brown, Thermo Fisher Scientific has more than 25,000 pre-made TaqMan miRNA assays in its portfolio, including all 2,500-plus human miRNAs from miRBase 21.
For those looking to quantify miRNAs directly from biofluids, there’s the Firefly assay from Abcam. According to Dan Pregibon, Abcam’s general manager for platform innovation, Firefly relies on a series of rod-shaped hydrogel microparticles. Each particle is barcoded and recognizes a specific miRNA; up to 70 barcodes can be combined in one sample.
As Pregibon explains, the sample is mixed with a pool of Firefly particles, each of which captures its complementary miRNA directly from a crude biofluid digest. Those miRNAs are amplified with biotinylated primers, recaptured on the particle and labeled with a streptavidin-conjugated fluorophore. Finally, the particles are passed through a flow cytometer, which reads out the identity of the miRNA being quantified and its abundance.
According to Pregibon, five premade Firefly panels are available (for oncology, immunology, neurology, cardiology and toxicology). Custom panels also are available.
Better cancer diagnosis
The hoped-for result of all these tools, of course, is new biomarkers and therapies that alter the course of cancer diagnosis and treatment. At least 20 miRNA-based clinical trials were ongoing as of 2014 [1], and more are surely in the pipeline. But miRNAs represent just one class of noncoding RNA, McGregor says, and the other classes are being shown to play key regulatory roles, too. McGregor, for one, is excited about the possibilities.
“Long noncoding RNAs, piwiRNAs, tRNAs—the whole noncoding RNA world is definitely one to watch,” she says.
Reference
[1] Hayes, J, Peruzzi, PP, Lawler, S, “MicroRNAs in cancer: Biomarkers, functions and therapy,” Trends Mol Med, 20:460-9, 2014. [PMID: 25027972]