title: 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.
authors: Suzhou Institute of Nano-tech and Nano-bionics, Chinese Academy of Sciences, 398 Ruoshui Road, Suzhou, 215123, China.
authors: Demin
authors: D
authors: Duan
authors: Ke-xiao
authors: KX
authors: Zheng
authors: Ye
authors: Y
authors: Shen
authors: Rong
authors: R
authors: Cao
authors: Li
authors: L
authors: Jiang
authors: Zhuoxuan
authors: Z
authors: Lu
authors: Xiyun
authors: X
authors: Yan
authors: Jiong
authors: J
authors: Li
mesh_terms: D020869:Gene Expression Profiling / Q000379:methods*; D057166:High-Throughput Screening Assays; D006801:Humans; D035683:MicroRNAs / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012313:RNA / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D035683:MicroRNAs; D012313:RNA