ABC Spotlight on emerging microRNA analysis methods

MicroRNAs (miRNAs) are short (~20-base) non-coding RNA sequences that are biochemical modulators of gene expression, influencing development, cell proliferation, apoptosis, and other processes [1]. As of 2013, nearly 2000 human miRNA sequences had already been identified [2]. Importantly, changes in the levels of miRNAs in many diseases, combined with the presence of these miRNAs in biological fluids (blood, urine, cerebrospinal fluid, etc.), make them promising biomarkers for a variety of diseases [1]. Leuenberger et al. [2] further described the potential of miRNAs as biomarkers in the fight against doping in athletics. Accurate determination of the concentrations of specific miRNA sequences in clinical and biological samples is thus important. However, miRNAs have a number of intrinsic characteristics, including low concentrations (pM to fM), short lengths, sequence similarity, and difficulty in selective amplification, all of which make the accurate quantitation of miRNAs challenging [1]. Interestingly, the most prevalent method for studying miRNA, quantitative reverse-transcription stem-loop PCR, is somewhat of a misnomer because it does not actually quantify miRNA concentration. Instead, it determines the cycle of quantitation (Cq), or the number of thermal cycles in PCR needed to confirm specific amplification. Thus, relative values such as Cq or related measures for miRNAs, instead of actual concentrations, are typically found in the literature. Clearly, the development of improved methods for measuring the concentrations of miRNAs in clinical and biological specimens is an important challenge for the bioanalytical chemistry community. This Spotlight focuses on recent advances in miRNA analysis, including sample preparation methods and detection approaches. A key challenge in the detection of miRNAs is sample preparation because miRNA represents a very small part of the RNA content in biological samples; for example, miRNA comprises about 0.01% of the total RNA in cells [3]. A 2-fold increase in the quantity of miRNA recovered from cells was reported when a nonionic surfactant (Triton X-100) was used in conjunction with the standard detergents in a commercial miRNA preparation system (TRIzol, Life Technologies, Grand Island, NY) [3]. The improved miRNA extraction was verified through capillary electrophoresis analysis of miRNA-21 extracted from H9c2 and HeLa cells. In body fluid samples, such as urine or blood, miRNA is found in exosomes, lipid bilayer vesicles that are usually <100 nm in diameter and are transferred from cells into the extracellular space. Exosomes are typically purified through differential ultracentrifugation, which can be both time consuming and labor intensive, such that improved exosome preparation strategies are of interest. Gale et al. [4] recently demonstrated the use of field flow fractionation methods to separate exosomes derived frommouse melanoma cultured cells. They found that exosome subpopulations could be separated without immunoaffinity or other labeling steps that are typically used but complicate the process. Field flow fractionation is also an approach that can be adapted for the preparative scale, potentially improving throughput and reducing the time needed to collect exosome fractions. Because of challenges associated with the quantitation of miRNA, numerous strategies are being pursued to provide sequence-selective and simple quantitation of miRNA. One emerging approach involves sequence-specific hybridization of miRNA to a complementary locked nucleic acid oligonucleotide probe, followed by treatment with poly(U) * Adam T. Woolley atw@byu.edu