MicroRNAs Are Emerging as Diagnostic Tools

Testing biologic samples against a hybridization array that contains a library of microRNAs enables construction of a microRNA expression profile for specific biologic states. If the sample is from a tumor, the identity of microRNAs present in the tumor tissue can be used to learn about cancer mechanisms, to gain prognostic or diagnostic information, or to search for potential biomarkers.

One of the pioneers in the development of microRNA-based products, Rosetta Genomics Laboratories (Rehovot, Israel) developed technologies that enable identification of microRNA sequences in genomes of a wide range of sample types. Its platform technologies help to identify, extract, quantify, and analyze microRNAs, and to measure their expression level. These technologies have enabled the advancement of multiple diagnostic projects that address unmet needs in cancer, women's health, and other indications.

Tests produced by Rosetta include miRview mets, a test that can accurately identify the primary tumor site in patients presenting with metastatic cancer, as well in patients whose tumor has not been identified; miRview squamous, which differentiates squamous from nonsquamous, non-small cell lung cancer (NSCLC) patients; and miRview meso--a test that leverages microRNA's high specificity as biomarkers to differentiate mesothelioma, a cancer connected to asbestos exposure, from other carcinomas in the lung.

Miltenyi Biotech's (Gladbach, Germany) microRNA microarray product is called 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.

miRXplore a product of Miltenyi, has been used in expression profiling of blood-derived cells. Control oligonucleotides help to compensate for bias and allow reliable data normalization. Many users of this product are immunologists.

Miltenyi uses an optimized buffer that reduces melting temperature differences between adenine-thymine (AT) and guanine-cytosine (GC) base pairings. It permits sensitive and specific detection of all microRNAs simultaneously.

To distinguish microRNAs that differ by as little as one nucleic acid (NA) is a challenge. Miltenyi performed experiments with artificial mismatch microRNAs 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. Therefore miRXplore can distinguish between microRNA family members that have a high degree of sequence similarity.

Exiqon (Tustin, CA, USA) offers miRcury locked nucleic acid (LNA) arrays for expression profiling. The LNA technology uses NA 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 the 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.

Quantitative polymerase chain reaction (qPCR) is also used for expression profiling of microRNAs. Biotrove (Woburn, MA, USA) offers OpenArray for medium to high throughput qPCR, and its data loss prevention (DLP) product for microRNA expression profiling. The technology in the OpenArray plates allows 2,688 TaqMan qPCR reactions to be done in parallel. More than 30,000 data points can be collected in one day. The platform is flexible enough to carry out screening projects.

Biotrove assembles the OpenArray DLP assays with its customer's primers into the wells of the plate. The customer then loads the 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.

miRNA profiling experiments work in cancer and cardiovascular research, but the OpenArray platform is also widely used in microbial detection and gene expression markets.

One item waiting to be developed is a microRNA expression profiling system that will profile microRNAs and messenger (m)RNAs at the same time, and then extract information about gene networks and functional systems. The problem that needs to be overcome is the different conditions necessary for microRNA and mRNA capture.

Results indicate that microRNAs are superior to other biomolecules for biomarker discovery purposes. The mechanistic information revealed by microRNA expression profiles might solve, in the future, numerous difficult biologic problems.

Related Links:
Rosetta Genomics
Miltenyi Biotech
Exiqon
Biotrove