Advancements in health research.
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Advancements in health research.
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Introduction. The pathophysiology of neuropathic pain remains only partially understood. There is a growing interest about altered gene and protein expression underlying this pathologic entity. (1) Small fiber neuropathy (SFN) is a neuropathic pain condition characterized by selective impairment of small-diameter A-delta and C-fibers. Diagnosis relies on clinical characteristics and assessment of intraepidermal nerve fiber density (IENFD). (2) MicroRNAs (miRNAs), non-coding regulatory RNAs, have emerged as biological regulators modulating gene expression related to pain sensitization. (3) The aim of this study is to investigate the expression a subset of miRNA as part of the pathomechanism underlying chronic pain in patients with SFN, and to correlate their abundance with the IENFD, in order to identify molecular signatures associated to these characteristics.Methods. A literature review was conducted to establish scientific rationale for investigation of altered miRNA expression in painful polyneuropathy patients, identifying candidate for expression analysis: miR-146a-5p, miR-155-5p, miR-203a-3p, and miR-21-5p. For the quantitative analysis we selected 16 patients with a diagnosis of SFN: 8 of them showing denervated pattern (“Den”) at skin biopsy and 8, age and sex-matched, with normal (“Norm”) innervation pattern, all recruited at Fondazione IRCCS Istituto Neurologico C. Besta of Milan, Italy. Total RNA was isolated from the epidermis of two 50-um sections per subject, after tissue dissecting under the microscope, using PureLink FFPE Total RNA Isolation Kit (Invitrogen). (4) All RNA samples achieved adequate purity ratios (A260/A280 = 1.4–2.4) and concentration (1.6-6.6 ng/uL). The expression levels of four selected miRNAs were quantified using Digital Droplet PCR (ddPCR) and the values obtained were normalized with ubiquitarian and invariant non-coding RNA sequences SNORD44 and SNORD48 as endogenous controls. (8) Statistical analysis has been conducted with Kolmogorov-Smirnov testing, Mann-Whitney U tests for group comparisons, and Spearman's correlation for reference endogenous control assessment, with significance set at p
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