MicroRNA-derived Fragment Length Polymorphism Assay

Abstract

MicroRNA (miRNA) studies are experiencing a transition from basic research applications to clinical applications. However, the lack of reliable and sensitive miRNA detection methods has become a bottleneck in the process. Here, we report an absolute quantification method based on the competitive PCR amplification of specific miRNAs and synthetic RNA spike-ins in a single reaction. RNA spike-ins are quantified as dynamic RNA copy number standards and are used to measure selected miRNAs free from the effects of intra-assay variables, including those from individual sample sources. Combined with the size differentiation power of capillary electrophoresis, the content of miRNAs was reproducibly measured, with verifiable detection limits of 10–46 copies over 5-log detection ranges. The direct measurements of miRNAs from 168 human serum samples and their considerable value as a diagnostic for bronchopneumonia and bronchiolitis demonstrate the potential of the assay in clinical applications.

Use of a 6-miRNA panel to distinguish lymphoma from reactive lymphoid hyperplasia

“…The levels of the original miRNA panel in 124 lymphoma samples that covered the major lymphoma types and 20 RLH FFPE samples were measured using a microRNA-derived fragment-length polymorphism (miRFLP) assay, which is an extract-free single-reaction assay. 6 After normalization to let-7f levels, the differential expression of 11 miRNAs is shown in Fig. 1c ( Table S1).…”

The plasma miR-122 basal levels respond to circulating catecholamines in rats

“…administration planning. The plasma miRNAs (miR-122, miR-192, miR-92 and miR-451) were quantitated simultaneously by a multiplexed plasma-direct miRFLP assay as reported previously (Xie, et al 2015, Zhu, et al 2017.…”

“…We utilized modified plasma-direct miRFLP assay with noise-reduction adapters to quantitate the absolute concentration of miRNA from plasma samples directly. The detailed method was reported previously (Xie, et al 2015, Zhu, et al 2017. The absolute copy numbers of plasma miRNAs were calibrated against a serial dilution of miRXplore Universal Reference (Bissels, et al 2009).…”

“…Additionally, the diurnal variation of plasma miR-122 (Heegaard, et al 2016) is also a concern for its intraday variance, especially those during daytime. In an attempt to understand what contribute to the high variation of circulating miR-122 measurement, we utilized plasma-direct miRFLP assays (Xie, et al 2015, Zhu, et al 2017 and stress-trained rat models to explore the effects of stress and drugs on plasma miR-122 levels.…”

Small and Smaller—sRNAs and MicroRNAs in the Regulation of Toxin Gene Expression in Prokaryotic Cells: A Mini-Review

“…Up to 2014, 24,521 microRNAs had been discovered, and they are important components of many regulatory pathways [ 45 ]. Although such small molecules have been found broadly in multicellular organisms, there is an opinion that typical prokaryotic microRNAs do not occur [ 46 ]. Interestingly, a few microRNA-size small RNA fragments (15–26 nt) have recently been reported in prokaryotic cells.…”