Label-free high-throughput microRNA expression profiling from total RNA

Abstract

MicroRNAs (miRNAs) are key biological regulators and promising disease markers whose detection technologies hold great potentials in advancing fundamental research and medical diagnostics. Currently, miRNAs in biological samples have to be labeled before being applied to most high-throughput assays. Although effective, these labeling-based approaches are usually labor-intensive, time-consuming and liable to bias. Besides, the cross-hybridization of co-existing miRNA precursors (pre-miRNAs) is not adequately addressed in most assays that use total RNA as input. Here, we present a hybridization-triggered fluorescence strategy for label-free, microarray-based high-throughput miRNA expression profiling. The total RNA is directly applied to the microarray with a short fluorophore-linked oligonucleotide Universal Tag which can be selectively captured by the target-bound probes via base-stacking effects. This Stacking-Hybridized Universal Tag (SHUT) assay has been successfully used to analyze as little as 100 ng total RNA from human tissues, and found to be highly specific to homogenous miRNAs. Superb discrimination toward single-base mismatch at the 5′ or 3′ end has been demonstrated. Importantly, the pre-miRNAs generated negligible signals, validating the direct use of total RNA.

5-Fluorouracil in combination with cisplatin alters the microRNA expression profile in the CNE nasopharyngeal carcinoma cell line

“…The SHUT assay, recently presented by Duan et al (23), is an efficient technique for high-throughput miRNA profiling using microarrays. Being a label-free approach, it eliminates the cost and potential biases of the fluorescent-labeling of miRNAs, while offering good specificity and sensitivity.…”

“…The raw pixel intensities were extracted using the GenePix Pro 7.0 software (Molecular Devices, Inc.). Data processing (including filtration, background-correction, transformation and normalization) and subsequent statistical analysis were performed using the methods provided by Duan et al (23). Finally, the expression levels of all mature miRNAs were plotted on a heatmap.…”

“…In this study, we used a label-free highthroughput miRNA array technology, the stacking-hybridized universal tag (SHUT) assay (23), to investigate the effect of cisplatin and/or 5-FU exposure on the global expression profile of miRNAs in the CNE human NPC cell line. 10 ) values were calculated.…”

Coupling hybridization chain reaction with catalytic hairpin assembly enables non-enzymatic and sensitive fluorescent detection of microRNA cancer biomarkers

“…However, the method has some limitations such as the requirement of isolated and highly purified total RNA samples which are difficult to obtain and the short length of the miRNA which has restricted the direct application of the conventional RT-PCR protocols (Chen et al, 2005). The shortcomings of the traditional methods have thus intrigued the development of new signal amplification approaches for sensitive and selective detection of the miRNA biomarkers (Jia et al, 2010;Duan et al, 2011). To achieve this goal, a number of signal amplification strategies employing nanomaterial (Fang et al, 2006;Alhasan et al, 2012) or enzyme (Duan et al, 2013;Yu et al, 2013) levels of miRNAs.…”

Rapid Sub-attomole MicroRNA Detection on a Portable Microfluidic Chip

“…However, its reliability is relatively low [20][21][22][23] and, as well as the other technologies, it requires running time of at least a few hours to overnight. This is because the incubation time is limited by the free diffusion of sample molecules during surface hybridization.…”