By Bernard Tulsi

Vincent Pallotta loads an RNA sample onto Ambion's flashPAGE Instrument, which is used to isolate mature microRNA or other small nucleic acids.
With the emergence of RNA interference (RNAi) as a powerful analytical tool in mammalian genetic screening, the discovery of strong correlations between the 217 known microRNAs (miRNAs) in the development and differentiation of tumors, and the potential to develop diagnostics based on this finding, the RNA research field has seen a marked resurgence in its vitality.

The ability of RNAi to silence genes has already earned it a place among the most important advances in biomedical research. In recognition, the Broad Institute, for example, has launched a platform to harness the potential of RNAi in identifying links between genes and disease processes as well as to illuminate underlying biological mechanisms. The institute’s RNAi platform, which emerged from The RNAi Consortium (TRC), a collaboration of six academic research institutions and five pioneering life sciences organizations, is focused on developing materials and technology to facilitate RNAi applications in mammalian genetic screening.

Meanwhile, a research team from the University of Massachusetts Medical School reported (April 24 issue of Nature Structural & Molecular Biology journal) what could amount to a significant addition to the RNAi armamentarium, that is a method to silence some of the hard-to-knock-down genes. In essence, their approach rendered targeted genes more vulnerable to silencing by raising the accessibility of their messages to RNAi tools, thereby boosting the silencing effect from 25% to 65%.

“Since 2001, when more was learnt about the applicability of RNAi in mammalian cells, there has been an explosion of applications in target identification and validation,” says Craig Mickanin, PhD, Research Investigator, Functional Genomics, Novartis Institute of Biomedical Research. The maturation of RNAi allowed many groups to initiate efforts to create very large, even genome-scale reagent collections to do saturation loss of function studies, says Mickanin.

Matching enthusiasm has been ignited in the miRNA field as well. It is emerging out of a sleepy period that started when the tiny RNA molecules were first discovered in 1993 in the nematode C. elegans, when they were considered a novelty, or even a biological oddity. A lot of the foundation work has been done by James Carrington, professor and director of the Oregon State University Center for Gene Research and Biotechnology and his team. Carrington believes “In molecular biology, micro-RNAs are clearly one of the top two or three discoveries of the past decade."

Interest started to build in 2001 when three labs separately published findings indicating several hundred miRNA genes in the human genome based on random cloning and sequencing experiments. By 2002, the direction started to shift from merely identifying those genes to showing their involvement in cancer. Publications that year pointed to a possible involvement of miRNA in leukemia and related areas.The interest in cancer has persisted. “The excitement is now centered on the potential that miRNAs are involved with the developmental steps related to a cell becoming part of a cancerous lineage, or oncogenesis,” says Jim Jacobson, PhD, VP, Research & Development, Luminex Corp.

David Brown, PhD, Associate Director of R & D, Ambion, Inc. concurs. “The field has gone in the past three years from this very nascent examination of inhibitory RNAs to now identifying and understanding them as key regulators of gene expression that undoubtedly have profound effects on human health — that’s where the field is today.

“It is being recognized that misregulation and mutation of these small RNAs can have profound effects on proper development and differentiation. That is what is driving the field right now — there is great interest in what roles miRNA might have in systems and diseases, whether they may provide some analytes for diagnostics or even some targets for therapeutics.”

Kiet Tran resuspends RNA
oligonucleotides using one of Ambion’s automation instruments.

miRNA and cancer

In the June 9 issue of Nature, researchers from the Broad Institute of MIT and Harvard, the Dana-Farber Cancer Institute, MIT, and St. Jude's Children's Research Hospital in Memphis, TN, reported two important breakthroughs: “a surprisingly accurate correlation of the 217 known human microRNAs (miRNAs — small non-coding RNA molecules that control the levels of proteins made from transcribed genes) with the development and differentiation of tumors, and the development of a technology that not only enabled this exciting discovery but that could be the basis for an easy and inexpensive diagnostic test.”

"Since the discovery that microRNAs control specific cell divisions in the nematode C. elegans, I have wondered if there might be a relationship between microRNAs and human cancer," said H. Robert Horvitz, co-author and David H. Koch Professor of Biology at MIT and HHMI investigator at Harvard.

"This work establishes a striking correlation between patterns of microRNA expression and cancer and offers the prospect of using microRNA expression patterns to help in the diagnosis and treatment of cancer."

A Broad Institute statement noted that to determine the expression pattern of all the known human miRNAs, the researchers first had to develop an accurate, fast, reproducible and inexpensive method. “Given the small size of miRNA (˜21 nucleotides) as well as their close resemblance to each other, previous attempts to use array-type technologies have been unsuccessful. Instead, the scientists developed an ingenious bead-based miRNA capture method, where each individual bead was marked with fluorescence "tags" that could tell which miRNA was bound as well as its abundance in the sample.”

Playing an important enabling role in this study was the xMap technology platform, which was developed by Luminex Corp. to address the need for multiplex testing, that is, analyzing more than two targets at a time, according to Jacobson. Such simultaneous measurement is a key breakthrough.

xMap is capable of running up to 100 different analytes simultaneously. “It is based on the use of very small polystyrene beads or microtubes, which are dyed with two different levels of fluorescent dyes.

“By using these two dyes at 10 concentrations each in combination with one another, we produce 100 different bead tests that are distinguishable from each other by their fluorescent characteristics.” The actual reaction takes place on the surface of the beads.

Jacobson explains that in the case of the Broad Institute study, the researchers placed a capture molecule on the surface of the beads to enable the detection of different miRNA species in their studies.

“They allow the reaction to take place and the target material is labeled with a third fluorescent material, called a reporter molecule. As the active beads pass through the analyzer, the Luminex 100, there are three kinds of fluorescence — the two that are inside tell what the investigators are looking for and the third bead tells how much of a reaction took place on that particular bead.”

Jacobson notes that this process can handle 96 samples in about 30 minutes. “There are about 2,700 of our systems in the market, more or less evenly distributed between bioscience research and clinical diagnostics. There are about five or six companies that have FDA approved applications on the platform and there are more in the pipeline.”

Microsphere-based xMAP platform (Luminex) offers focused multiplexing of 2-10 analytes for use in laboratories
performing proteomics and genomics applications.

Lingering technical challenges

Researchers in this field acknowledge a dearth in technologies to study miRNA. One of the most readily recognized limitations now is the inability of informatics technologies to keep pace with the data generating capabilities of the multiplexing systems.

To be sure, the technological challenges are older and more far reaching. “There were no effective methods for isolating miRNAs when we started two and a half years ago, and that is really the most basic component of doing any research project, being able to recover small RNA molecules from your sample,” says Ambion’s David Brown.

To get started, Ambion had to develop technologies to study expression of miRNA samples and compare them between samples. The company also focused its early technology development on the ability to do functional studies on miRNAs, “where we could introduce them into cells and ask what happened or eliminate them from cells and ask what happened,” says Brown.

Ambion has come a long way from those early days. As Brown contemplates future challenges, he notes that they key enabling technology will be the amplification of miRNA to facilitate array experiments on small miRNA samples. “This is probably the key remaining opportunity for technological development.”

Brown believes that over the next three or four years a lot of discoveries will relate miRNA to specific human diseases and specific biological processes. “What seems apparent now is that there are between 500 and 1,000 miRNAs and that they regulate the expression of between 50% and 75% of human genes, and when you think about the magnitude of that regulation, they are going to have profound implications for human medicine and human health,” hesays.

Dharmacon Scientist Dave Mierzejewski loads columns on to an RNA synthesizer.

RNAi applications

Mickanin’s group functions as an RNAi platform technology within Novartis Biomedical Research, and works actively with groups focusing on disease areas throughout the research organization.

“We work with them to set up cell based models of the physiological processes they want to analyze or for identifying novel targets for drug discovery. Those assays will then be optimized for large scale screening using our reagent collections.

“These will then go into a series of confirmation and secondary assay development, from which we hope to deliver a small number of highly curated potential targets for drug discovery to the group, and from which they will pursue drug development based on their interest and expertise.”

Target validation entails the functional inactivation of a given protein, and the methods used previously to accomplish that were quite laborious. With wider use of RNAi technology, Mickanin believes, “Large scale screening will become much more common place in the field and increase over the years. The quality of reagents will become much better and this will become a staple in the target validation and gene identification processes in the future.”