Vol.:(0123456789) 1 3 Pediatric Surgery International https://doi.org/10.1007/s00383-019-04558-2 ORIGINAL ARTICLE First steps in the development of a liquid biopsy in situ hybridization protocol to determine circular RNA biomarkers in rat biofluids Eimear Kirby 1,2,3 · Wai Hei Tse 2,3 · Daywin Patel 2,3 · Richard Keijzer 2,3 Accepted: 12 September 2019 © Springer-Verlag GmbH Germany, part of Springer Nature 2019 Abstract Purpose Epigenetic factors are involved in the pathogenesis of congenital diaphragmatic hernia (CDH). Circular RNAs (circRNAs) are epigenetic regulators amenable to biomarker profiling. Here, we aimed to develop a liquid biopsy protocol to detect pathognomonic circRNA changes in biofluids. Methods Our protocol is adapted from the existing BaseScope™ in situ hybridization technique. Rat biofluids were fixed in a gelatin-coated 96-well plate with formalin. Probes were designed to target circRNAs with significant fold change in nitrofen-induced CDH. FastRED fluorescence was assessed using a plate reader and confirmed with confocal microscopy. We tested maternal serum and amniotic fluid samples from control and nitrofen-treated rats. Results We detected circRNAs in rat serum and amniotic fluid from control and CDH (nitrofen-treated) rats using fluores- cent readout. CircRNA signal was observed in fixed biofluids as fluorescent punctate foci under confocal laser scanning microscopy. This was confirmed by comparison to BaseScope™ lung tissue sections. Signal was concentration dependent and DNase resistant. Conclusion We successfully adapted BaseScope™ to detect circRNAs in rat biofluids: serum and amniotic fluid. We detected signal from probes targeted to circRNAs that are dysregulated in rat CDH. This work establishes the preliminary feasibility of circRNA detection in prenatal diagnostics. Keywords Congenital diaphragmatic hernia · Circular RNA · Biomarker · Liquid biopsy · BaseScope™ Introduction Congenital diaphragmatic hernia (CDH) is a developmental birth defect which complicates 2.3–2.8 per 10,000 live births [1–4]. It is among the most common congenital anomalies reported [3], and represents a significant public health issue and economic burden [5, 6]. Early diagnosis of CDH enables multidisciplinary antenatal counselling, delivery planning, and identification of cases suitable for in utero therapeutic intervention. CDH lacks a definitive, biomarker-based diag- nostic and prognostic test. Accurate and scalable diagnostic tests are, therefore, imperative to identify CDH pregnancies in early gestation, and redirect goals of care as necessary. The inability to identify a common genetic etiology in the majority of CDH cases [4, 7] has led to the hypothesis that epigenetic factors play a role. We have previously published that microRNAs are downregulated in abnormal lung devel- opment and CDH [8], and that prenatal microRNA therapy improves lung development in nitrofen-induced CDH and reduces the incidence of CDH [9]. Circular RNA (circRNA) species can regulate microRNAs and have thus emerged as candidate biomarkers. CircRNAs are covalently closed, non- polyadenylated, single-stranded RNA structures which act as powerful epigenetic regulators. They arise as a result of head-to-tail “back-splicing” events, in which the 5′ end of an upstream splice donor site is joined to the 3′ end of a downstream splice acceptor site. Various properties of cir- cRNAs suggest that they are amenable to biomarker profil- ing: they are stable, conserved, enriched in biofluid samples, * Richard Keijzer richard.keijzer@umanitoba.ca 1 Trinity Biomedical Sciences Institute, Trinity College Dublin School of Medicine, Dublin, Ireland 2 Division of Pediatric Surgery, Departments of Surgery, Pediatrics & Child Health and Physiology & Pathophysiology, University of Manitoba, Winnipeg, MB, Canada 3 Children’s Hospital Research Institute of Manitoba, Winnipeg, MB, Canada