It begins with the introduction of the overall concept and strategies of miRNA expression detection methods emphasizing the need of a wide variety of these methods to suit specific requirements for research and clinical examination in laboratories. In the following, each single chapter focuses on an independent, unique method of miRNA detection and is divided into five subsections: summary, introduction, protocol (including materials, instrument, reagent, and procedure), application and limitation, and reference. The development of the technique, the ideas behind it and the mechanisms underlying the method are given in the introduction of each chapter. The step-by-step protocols are detailed in the protocol section. Then, the applications and limitations of the methods are discussed. Finally, the literature citations are listed in reference section. Schematic diagrams are included where needed and appropriate for better illustrating the principle of the methodologies. In addition, flowcharts are also provided to outline the protocols for each of the miRNA expression detection methods.
FIG. 1 shows a pictorial representation of one embodiment of the miRNA detection method of the present disclosure. In this embodiment, the capture oligonucleotides are coupled to a solid, internally color-coded microsphere (which serves as the substrate and contains the first signal tag).
In the normal course of cell growth and tissue differentiation, the RNA-induced silencing complex (RISC) is responsible for miRNA-mediated silencing of target mRNAs. But what if this complex could also serve as a tool to detect miRNA expression? That's the question Yoo et al. tackle in a brief communication in . There's no shortage of miRNA detection methods, most notably RT-PCR or deep sequencing. For studying miRNAs in cells, the most common approach is in situ hybridization of fixed cells or tissue sections. However, Yoo et al. were interested in studying intact, live cells, and they knew that signal strength would be a concern even as they sought to keep assay cost low. With these criteria in mind, they decided to use a sensor RNA oligo as a substrate for RISC. The oligo is fully complementary to the miRNA to be detected, pushing RISC toward target degradation. At one end of the oligo is a fluorescent dye, at the other a quencher. When matching miRNA is present, RISC cleaves the sensor oligo, unquenching the dye. Since RISC is then free to cleave other substrate oligos, signal amplification occurs. To test the strategy, the authors probed a breast cancer cell line with an oligo directed against miR-10b, which is believed to have a role in metastasis. Cells were first transfected with locked nucleic acid (LNA) antisense oligos either targeted against miR-10b or containing a scrambled sequence. Sensor oligos modified with 5′ Cy5 and a 3′ Iowa Black RQ were then added. Fluorescence in the wells in which miR-10b had been inhibited by the LNA oligo rose slowly over time, but was significantly lower than in the cells treated with scrambled LNAs, in which fluorescence peaked at 16 hours. Similar behavior was seen in a breast adenocarcinoma cell line. Assay readout, whether by epifluorescence or flow cytometry, agreed well with RT-PCR results. The authors appropriately characterize their results as an early feasibility study that will require further optimization. However, they conclude that their approach will be a low-cost, high-throughput method to detect miRNA in intact cells, enabling a more thorough investigation of the dynamics of this regulatory system.
This book summarizes microRNA (miRNA) biology in a variety of pathological processes, emphasizing the significant potential applications of miRNA in diagnostics and prognostics, as well as novel drug targets. The conventional techniques used for miRNA detection including standard PCR, Northern blotting, microarray and clone methods are addressed. Recent emerging strategies in miRNA detection and quantification with superior flexibility and adaptability, such as novel molecular biological techniques and locked nucleic acid (LNA) modified probes, as well as nanotechnology-based approaches, are also included. The book also highlights the latest advances in clinical-related miRNA detection methods in living cells, circulating blood and tissue, such as in situ hybridization (ISH) and molecular imaging techniques, which are useful to elucidate the biogenesis and biological function of miRNAs in vivo. Finally, the respective advantages and drawbacks of various detection techniques in this fast-moving field are discussed, along with the challenges and promising new directions.