Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform

Abstract

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in a single reaction. The miRNA assay counts transcripts by directly imaging and digitally counting miRNA molecules that are labeled with color-coded fluorescent barcoded probe sets (a reporter probe and a capture probe). Barcodes are hybridized directly to mature miRNAs that have been elongated by ligating a unique oligonucleotide tag (miRtag) to the 3' end. Reverse transcription and amplification of the transcripts are not required. Reporter probes contain a sequence of six color positions populated using a combination of four fluorescent colors. The four colors over six positions are used to construct a gene-specific color barcode sequence. Post-hybridization processing is automated on a robotic prep station. After hybridization, the excess probes are washed away, and the tripartite structures (capture probe-miRNA-reporter probe) are fixed to a streptavidin-coated slide via the biotin-labeled capture probe. Imaging and barcode counting is done using a digital analyzer. The immobilized barcoded miRNAs are visualized and imaged using a microscope and camera, and the unique barcodes are decoded and counted. Data quality control (QC), normalization, and analysis are facilitated by a custom-designed data processing and analysis software that accompanies the assay software. The assay demonstrates high linearity over a broad range of expression, as well as high sensitivity. Sample and assay preparation does not involve enzymatic reactions, reverse transcription, or amplification; has few steps; and is largely automated, reducing investigator effects and resulting in high consistency and technical reproducibility. Here, we describe the application of this technology to identifying circulating miRNA perturbations in irritable bowel syndrome. Video LinkThe video component of this article can be found at

MicroRNA-16 inhibits the TLR4/NF-κB pathway and maintains tight junction integrity in irritable bowel syndrome with diarrhea

“…Emerging evidence has pointed out the involvement of miRNAs in modulating specific biological processes in IBS-D, which may be translated clinically to restore intestinal functions and alleviate gastrointestinal symptoms (17,18). This present study revealed that miR-16 was under-expressed in the IBS-D patients and mouse model.…”

Global research states and trends of micro RNA in irritable bowel syndrome: a bibliometric analysis

“…Previous studies have identified miRNAs such as miR-29a/b, miR-24, miR-510, miR-212, miR-150, miR-342-3p, miR-199a, miR-125b-5p, miR-16, miR-144, miR-200a, miR-214, and miR-103 as potential therapeutic targets for IBS [ 80 , 81 ]. Simultaneously, some differentially expressed miRNAs have contributed to the diagnosis of different IBS subtypes [ 82 ]. Variations in transcriptomic profiles based on mRNA and miRNA may help distinguish UC from IBS [ 82 ].…”

“…Simultaneously, some differentially expressed miRNAs have contributed to the diagnosis of different IBS subtypes [ 82 ]. Variations in transcriptomic profiles based on mRNA and miRNA may help distinguish UC from IBS [ 82 ]. Additionally, miRNA therapy has several advantages.…”

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“…Furthermore, several miRNAs (miR-663b, miR-342, etc.) could be used to precisely distinguish the different subtypes of IBS ( 44 , 45 ). miRNAs exert a critical role in intestinal inflammation.…”