Editorial Article
Monday August 03, 2009
by Catherine Shaffer
MicroRNAs began primarily as an object of academic curiosity. Since their discovery, the scope and significance of microRNAs has expanded dramatically. They were associated with cancer early on, and have increasingly been connected to almost every area of disease and development. In fact, it would not be an exaggeration to say that microRNAs could be the key to nearly every unsolved problem in biology. MicroRNA expression is now an important part of the discovery process for a wide range of academic and commercial applications.
Capture Microarrays
The hybridization-based microRNA capture array is one of the most popular tools for characterizing a microRNA expression profile. The central repository for microRNA sequence information is the Sanger miRBase. By testing biological samples against a hybridization array that contains a library of microRNAs from miRBase or elsewhere, a microRNA expression profile can be constructed for a specific biological state. For example, if the sample is from a tumor, the identity of microRNAs present in the tumor tissue can be used to learn more about the mechanisms of cancer, to search for potential biomarkers, to gain prognostic or diagnostic information, and more.
Exiqon offers a set of microRNA microarrays for expression profiling, the miRCURY LNA™ Arrays. Exiqon's LNA, or locked nucleic acid, technology uses nucleic acid analogs in which a methylene bridge locks the ribose ring between the 2' oxygen and the 4' carbon. LNAs can form standard Watson-Crick base pairs, but with much greater affinity than natural bases. This increases sensitivity and specificity in microarray screening, and minimizes hybridization bias that tends to occur with the use of RNA or DNA capture probes.Version 11.0 of the miRcury Exiqon array contains more than 1,700 capture probes, including all microRNAs annotated in miRBase 11.0, viral microRNAs associated with these species, and an additional 428 miRPlus™ capture probes for microRNAs that are not annotated in miRBase 11.0. Since inception, roughly 30% of miRPlus™ sequences have been inducted into subsequent versions of miRBase. Says Chris Harbert, director of marketing, North American Life Sciences, Exiqon, "The validation steps they go through are similar to the validation steps required to be accepted into miRBase. Our miRPlus™ capture probes put our customers at the cutting edge of microRNA research. The target microRNAs are not annotated yet, not public knowledge, but [the Exiqon customers] get a first look at them."
A special challenge for microRNA hybridization is the wide range of melting temperatures of these small RNAs. It is difficult to define hybridization conditions at which all microRNAs can be detected with sufficient specificity and sensitivity.
Exiqon uses LNA technology to solve this issue. Another strategy for evening out this challenge comes from Miltenyi Biotec. Miltenyi uses a proprietary, optimized buffer that reduces melting temperature differences between AT and GC base pairings, which allows sensitive and specific detection of all microRNAs at the same time.
Miltenyi's microRNA microarray product is the miRXplore Microarray. It contains roughly 2,200 microRNAs covering human, mouse, rat, and virus, matching annotated microRNAs from miRBase 13.0 as well as 72 controls. Included spike-in control oligonucleotides help to compensate for bias and allow reliable data normalization. miRXplore has been used heavily in expression profiling of blood derived cells. Many of Miltenyi's customers come from the field of immunology. Says Simon Mauch, global marketing manager for Miltenyi, "MicroRNAs seem to be good diagnostic and prognostic markers for a lot of immunology diseases. MicroRNAs are definitely better diagnostic and prognostic markers than mRNAs."
Another challenge in microRNA expression profiling is distinguishing microRNAs that differ by as little as one nucleic acid. Miltenyi performed experiments with artificial mismatch microRNAs that they spiked into total RNA from hepatocellular carcinoma cells, glioblastoma cells, hippocampus cells, and CD4+ T cells to illustrate relative signal intensities of single or double mismatch probes. The results showed that cross-hybridization signals were less than 10%, even in the case of single base mismatches. As a consequence, miRXplore distinguishes reliably between microRNA "family members" that have a high degree of sequence similarity.
qPCR
Quantitative PCR (qPCR) can also be used for expression profling of microRNAs. Biotrove offers a system called OpenArray for medium to high throughput qPCR, and their DLP product can be used for microRNA expression profiling. The proprietary technology in the OpenArray plates allows the customer to do 2,688 TaqMan® qPCR reactions in parallel. More than 30,000 data points can be collected in one day. The platform is flexible enough to carry a project from discovery phase to screening. Says Andrew Bond, PhD, "For microRNA profiling, our system is perfect, in that the full set of human microRNAs can be looked at in triplicate on just one OpenArray. After the discovery phase, a customer can order new plates containing assays for just a handful of miRNAs, and they can easily switch to a screening mode, running through many samples per day."
Biotrove assembles the OpenArray DLP plates for the customer by loading the assays with the customer's primers into the holes of the plate. The customer then loads their samples and runs the plates on the OpenArray NT cycler. Each OpenArray DLP plate can run up to 48 separate samples, and the OpenArray NT cycler can handle three plates per run and four runs in a day, for a total of 576 samples in one day.
Customers running miRNA profiling experiments are working in cancer and cardiovascular research, but the OpenArray platform is also widely used in the microbial detection and gene expression markets. Says Dr. Bond, "I think it's exciting that researchers can purchase the full set of microRNA qPCR assays and then run them with the OpenArray platform. Our system will save them considerable time compared to everything else out there for miRNA profiling."
One item on many scientists' wish list is a microRNA expression profiling system that will profile microRNAs and mRNAs at the same time, and then extract information about gene networks and functional systems. The problem with this concept is that the conditions for microRNA and message RNA capture are different. Says Exiqon's Chris Harbert, "Trying to put them all in one box usually results in a compromise on one end of the scale or the other. Current microRNA arrays can't do it." Miltenyi has this target in their crosshairs when they perform genomics services for their customers by employing the capabilities that their bioinformatics group can offer. Miltenyi also works on development projects that would give a better analysis of the microRNA maturation pathway, from premature microRNAs to mature RNAs.
MicroRNA expression profiling is an exciting, young field with a great deal of future growth potential. Early results indicate that microRNAs may be superior to other biomolecules for biomarker discovery purposes, and the mechanistic information revealed by microRNA expression profiles may crack more than a few very difficult biological nuts. A number of choices exist for microRNA analysis platforms. Since small differences in microRNA expression could be biologically significant, it is important to have unbiased quantitative results, and this should be a prime consideration in choosing the right platform technology.