ABC Spotlight on emerging microRNA analysis methods

作者: Adam T. Woolley

DOI: 10.1007/S00216-015-8808-X

关键词:

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

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