Highly sensitive and specific in situ detection of circular RNAs using the BaseScope assay

Recently, a new universal class of endogenous noncoding RNAs that is generated by “head-to-tail’ splicing in eukaryotes gained considerable interest. This alternative spicing event results in the covalent linkage of the 5’ end of one exon with the 3’ end of another exon and creates stable single-stranded circular RNA molecules (circRNAs) (Andreeva and Cooper 2015, Salzman et al. 2013, Salzman 2016). These highly conserved circRNAs are characterized by tissue- and developmental stage-specific expression patterns (Memczak et al. 2013). More specifically, it was recently shown that circRNAs are particularly enriched in the brain where they are often derived from genes that code for synaptic proteins and with expression dynamics independent of the linear mRNA transcripts (Rybak-Wolf et al. 2015, You et al. 2015). Although abundantly expressed in the nervous system, their function remains largely unknown. Also, evidence has emerged across different fields that pinpoints a role for circRNAs in various diseases, including cancer, possibly by regulating gene expression levels through interaction with other molecules (eg. miRNAs) (Memczak et al. 2013, Wang et al. 2016, Yu et al. 2016). Therefore, the accurate detection and localization of circRNAs is a key factor to elucidate their functions especially given the fact that they could serve as putative clinical biomarkers.

BaseScope^TM is a novel in situ hybridization technology that allows visualization of specific exon-exon and exon-(retained) intron junctions in a highly specific and sensitive manner in cells and tissues. Besides the robust identification of splice junctions, BaseScope^TM in situ hybridization also provides high resolution single-cell spatial information within the morphological context of the tissue. To illustrate the strength of this BaseScope^TM application for the intriguing field of circular RNAs, we show the specific anatomical localization and cellular distribution of 3 target probes for Dlgap1 detecting the circRNA splice variant, the linear mRNA counterpart, and total Dlgap1 RNA in normal mouse brain.

Figure 1. Probe design for circular RNA splice variant detection using BaseScope^TM technology. (A) General illustration of 3 target probes to specifically detect the circular RNA, linear mRNA and total RNA for a target gene of interest. (B) BaseScope^TM probe design for Dlgap1.

Figure 2. Highly sensitive and specific in situ detection of circular RNA, linear mRNA and total RNA for Dlgap1 in normal mouse brain hippocampus using BaseScope^TM assay. The FastRED BaseScope^TM assay allows for both chromogenic (left panels) and fluorescent (right panels) signal detection.