Direct detection and quantification of microRNAs

In this review we have covered several unique miRNA detection and quantification techniques that may be applied to direct or in situ screening assays. While these novel designs hold immense promise for the future of miRNA detection, there are still several issues which must be addressed before they are accepted among the current standard PCR or Northern blotting methods. Generally, the methods must be more adaptable. For instance, the molecular beacon design by Paiboonskuwong is currently limited to miRNA targets that are flanked by guanines in their primary transcripts. Without the guanine in appropriate proximity to the quencher, detection is not limited to the mature form of the target. In the methods developed by Gao and Fan, the efficiency of tagging of miRNAs with electrocatalytic moieties and nanoparticles must be further evaluated before their methods can be considered mainstream. Of equal concern is that many of the methods described in this review are not adaptable to multiplex miRNA detection platforms, which is essential for miRNA expression profiling. While many of the discussed assays demonstrated exceptional adaptability, the assay developed by Neely for instance (which tested 45 human miRNAs in 16 different tissues), the robustness of these methods must be further explored before they can be used as standard procedures. This is especially true of the solution-phase assays, which may be applied directly to a variety of complex matrices and hopefully display acceptable background tolerance. These are the kinds of challenges the discussed assays will face as they are further developed. As we continue to discover the vast impact that miRNAs have on the course of diseases, such as cancer, and the immense promise they hold in the area of gene therapy, rapid and sensitive methods for their direct detection and quantification are of utmost importance. A simple approach to direct detection is to monitor some type of hybridization event. However, the small size and low in situ concentration of miRNA molecules typically foist signal or target amplification as a prerequisite and make the direct detection approach less viable through procedural complication. To address these problems, novel techniques have been developed which employ one or more of the following: signal amplification through enzyme substrate turnover, highly favorable hybridization conditions using LNA or PNA capture probes, highly sensitive forms of spectroscopy such as FCS or bioluminescence, optimized reporter molecules such as gold nanoparticles, or highly attuned and responsive electrocatalytic/amperometric monitoring. These discussed methods stand out above the rest because they have achieved comparable or improved sensitivity and specificity with respect to the current "gold standards" without the necessity for potentially hazardous materials, such as radioactive labels or toxic dyes, and highly expensive imaging equipment. These factors are becoming increasingly important in today's environmentally conscious society, and by drastically reducing the procedural complexity and overall expense of such assays, it opens the doors to academic environments, making research in the field of miRNAs much more accessible. © 2009 Elsevier Inc. All rights reserved.