Novel method for the isolation and measurement of microRNA and microRNA related targeted genes in conjunctival epithelial cells in primary Sjogren’s syndrome patients

Abstract

AbstractPurpose Sjogren’s syndrome (SS) is a systemic autoimmune disorder characterized by inflammation that affects exocrine glands notably the salivary and lacrimal glands leading to sicca complex. Dry eye disease in primary SS (pSS) is a combination of reduced lacrimal flow from gland destruction and tear hyperosmolarity leading to inflammatory damage on the ocular surface. Several studies have shown differential expression of certain miRs in PBMCs and salivary glands from SS patients compared to healthy controls, however no functional role has been shown. This study aims to isolate miRs and mRNA from conjunctival epithelial cells by impression cytology to determine if dysregulated miR expression contributes disease pathogenesis in pSS.Methods Impression cytology (IC) was optimized using different membranes. miRs and mRNAs were isolated from conjunctival epithelial cells (CEC) obtained from pSS patients and healthy controls by IC and sent for screening to Ocean Ridge Biosciences. qPCR was performed to evaluate expression of miRs from CEC.Results Milicell biopore was the best membrane with significant yield of miRs (47.5ng ± 0.6) and mRNA (158.0 ng ± 21.6) as well as patient comfort. Appreciable levels of miRs and targeted genes were detected from the CEC from healthy controls, confirming that it is possible to isolate miRs and miRs targeted genes from CEC. Screening studies in CECs demonstrated differential expression of miRs and mRNA between pSS patients and healthy controls.Conclusion Future work will identify and confirm specific roles of these miRs for future diagnostics, prognostics and therapeutics of pSS patients as well as other ocular surface conditions.