Researchers studying microRNA biology and therapeutic potential choose their favorite flavor of microarray to do the job.
Once upon a time, microarrays came in only one flavor: expression arrays. And, although limited to just measuring gene expression from RNA in high throughput fashion, this was quite satisfying for most. However, for the non-complacent, one flavor of array just wasn’t going to cut it. So, array developers went back to the drawing board and, well, came up with some new flavors. Today, there are microarrays to measure gene expression, to detect single nucleotide polymorphisms, to detect copy number variation, to sequence whole genomes, to measure expression of microRNA (miRNA), and much more.
The study of miRNA is a fairly recent phenomenon. And, of course, the fact that these small RNAs have made such a big splash is no accident—they have been explored as possible therapeutic molecules, under the heading of RNAi. But, in addition to their popularity in the biopharmaceutical industry, miRNAs are also important to academic researchers. One academic researcher harnessing the power of microarray technology to study miRNAs is Chris Davis, PhD, a research associate in the Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology (VCAPP) at Washington State University (WSU), Pullman, Wash. Together with prominent sleep scientist Dr. James Krueger (also at VCAPP-WSU), Davis looks at the molecular biology of sleep regulation and circadian rhythms in rodent models. Since 2005, Davis has been using the mirVana microarray platform from Ambion/Applied Biosystems (Life Technologies Corporation) of Austin, Texas, to study changes in miRNA gene expression in multiple rat brain regions at different times of day or following sleep-deprivation.
After preserving tissues in RNAlater (Sigma-Aldrich, St. Louis, Mo.), total RNA was isolated, and then fractionated, using Ambion’s flashPAGE Fractionator, to yield an enriched miRNA fraction for subsequent microarray analysis. “From the brain extracts that we looked at, we were able to visualize somewhere between 100 and 150 miRNAs. The others were below detectable levels, at least in the brain,” says Davis. “The mirVana array library probed 640 miRNAs, of these the majority of human miRNAs were conserved in rodents, although there were several rat- and mouse-specific miRNAs. We were able to get about 50 miRNAs that changed with varying conditions of sleep propensity, and only a handful were specific to rodents. Using this array platform, we ultimately converged on a short list of candidate miRNAs that we believe are important intermediates in the regulation of sleep.”
In another case of miRNAs meet microarrays, Ping Jin, PhD, staff scientist, in the Department of Transfusion Medicine, the US National Institutes of Health, Bethesda, Md., uses a home-made microarray platform for her studies of miRNA expression in leukocyte stems cells. Printed on this custom array are more than 800 miRNAs of human, murine, and viral origin. For the last two years, Jin has been using this array to compare human miRNA expression profiles of two different lines of therapeutic stem cells. After isolating the cell lines using specific, cell-surface markers, RNA is extracted and subjected to microarray analysis.
The array developers
Agilent Technologies, Santa Clara, Calif., is a developer of microarrays for miRNA analysis. “The arrays were first commercialized in May, 2007. And, at that time, we had one offering, a human miRNA array,” says Sangita Parikh, MS, MBA, senior product manager at Agilent. “At that time, the Sanger database was at 9.1, and that was what we delivered the product on.”
Hui Wang, PhD, senior research scientist at Agilent, who led the technical development of the miRNA microarray, explains some of the goals that went into its design. “One goal was to have a very simple protocol to minimize the experimental variation that can occur with any change in sample handling,” says Wang. “The second goal was to have a very direct measurement, meaning that we do not amplify the sample or size-fractionate, [which was also done] to minimize any alteration that can change the profile. And the third [goal] was to have very reproducible results.”
The arrays owe part of their high reproducibility to Agilent’s strong, custom probe design capability, which allows users to design long probes, with a length of 60 nucleotides, for a variety of DNA and RNA profiling needs. The probe design for miRNAs contains a hairpin structure that directly abuts the hybridized target miRNA. “This hairpin can not only help stabilize the hybridized target miRNA by stacking energy, but it also has the added benefit of discouraging the binding of RNAs that have very similar sequence to the intended target miRNA … ,” Wang explains.
With the high sequence homology between miRNA species, cross-hybridization in miRNA arrays is always a challenge to be overcome. However, by carefully and empirically determining the melting temperature of their array probes, Agilent is able to overcome this cross-hybridization problem. “We actually generate melting curves for a lot of our probes and choose the optimal probe sequences to balance the maximum signal versus the maximum specificity,” explains Wang. “The more stable the probe is, the greater the probability that it will cross-hybridize with highly-homologous targets; usually a single nucleotide mismatch is a deal-breaker for this interaction on the Agilent platform. And, to make sure we have the maximal sensitivity, all of the probe-target interactions are just above the melting temperature.”
The labelers
Mirus Bio, Madison, Wisc., does not develop miRNA arrays. However, they have developed a chemical-labeling method to label miRNAs called Label IT miRNA Labeling Kits, Version 2. “From our perspective, some of the challenges with miRNA microarray analysis are that they’re not completely accurate, and the Label IT labeling method is one way to improve accuracy,” says Shannon Bruse, PhD, director of scientific operations at Mirus Bio. Bruse’s statement is based on a number of studies, performed by Mirus Bio, that showed differences in accuracy between chemical-labeling methods and some of the more common enzymatic-labeling methods. “Chemical-labeling allows you to see more of the miRNA profile that’s there because you label all miRNAs in an unbiased fashion; some of the enzymatic methods don’t do that.”
The chemical reagent molecule is composed of three parts. The first, the chemically-activating part, is an alkylating agent that forms a covalent attachment with any nucleic acid. The middle part of the molecule is positively-charged and helps facilitate electrostatic interaction between the labeling reagent and the negatively-charged nucleic acid. Finally, a fluor—typically Cy3, Cy5 or biotin—is conjugated to this reagent, allowing the nucleic acid interaction to be detected.
The method is not specific for miRNA. “This reagent labels any nucleic acid base, which is pretty important to miRNA because there are other methods of chemical labeling that are specific to particular bases,” says Bruse, who adds that if a miRNA molecule does not contain that specific base, it will remain unlabeled. In addition, Mirus’ method allows users to control the density of labeling (by adding enough reagent to label one in every 20 base-pairs of a nucleic acid sequence, for example), which can ensure that the typical 20-mer miRNA molecule is labeled.
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A new twist
A variation on the approach of measuring miRNA expression using a microarray is the qNPA ArrayPlate method, developed by High Throughput Genomics (HTG), Tuscon, Ariz. qNPA, which stands for quantitative nuclease protection assay, is the first part of the miRNA expression analysis. In this assay, RNA is prepared and then hybridized with target-specific, custom-designed oligonucleotide probes (which HTG refers to as nuclease protection probes) to form DNA-RNA heteroduplexes. S1 nuclease, which digests single-stranded nucleic acid, but not double-stranded nucleic acid, is then added to the sample to produce a library of shorter, double-stranded heteroduplexes; the nuclease is then inactivated. In the second step of miRNA expression analysis, the library of heteroduplexes is first treated to remove the RNA portion, leaving the protected portion of the nuclease protection (DNA) probe intact. Then, this probe is hybridized, via a program linker, to 16 unique anchors on HTG’s universal ArrayPlate technology. What follows is a series of linker-based, hybridization steps that culminate in the construction of a detection complex; detection is based on the enzymatic activity of horseradish peroxidase.
This new custom array has only been on the market since October, 2008, and pharma scientists are already lined up to use it to answer questions such as: Does a miRNA of interest have adverse cytotoxic effects? “About 800 miRNAs will be plated onto a 3D surface, where you will be able to look at mouse, human, and rat miRNAs that have currently been discovered. We have a few folks in pharma and a few folks in academia that are very interested in these sets of miRNA,” says Billie-Jo Kerns, vice president of strategic marketing and business development at HTG. In addition, this high throughput array can be used to interrogate miRNA and messenger RNA expression, simultaneously, from the same sample; probing for mRNA of house-keeping genes allows the user to normalize miRNA expression.
In summary, the study of miRNA biology is crucial to achieving a better understanding of how these molecules control gene expression in the cell. Luckily, there is the miRNA flavor of microarray technology to greatly aid in the measurement of miRNA gene expression. Future development of this technology will enable researchers to discover novel miRNAs, understand miRNA biology, and solidify RNAi therapeutic applications. And, who knows, maybe a new flavor will emerge.
Published in Drug Discovery & Development magazine: Vol. 12, No. 1, January, 2009, pp.28-30.
Filed Under: Genomics/Proteomics
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