LNA-Probe-Based MicroRNA In Situ Hybridization in Brain Organoids.

MicroRNAs (miRNAs) are short regulatory RNAs that take part in posttranscriptional regulation. Their mature and active form is limited to 18-24 nucleotides and therefore sets demands on probe design for specific detection by in situ hybridization (ISH) analysis. Locked nucleic acids (LNA) are synthetic RNA analogs that augment binding affinity when incorporated into antisense oligos. In this ISH protocol, we applied LNA-enhanced probes in an automated set-up using a Ventana Discovery Ultra instrument for detection of miRNAs in formalin-fixed and paraffin embedded (FFPE) iPSC-derived brain organoids. In brain, several miRNAs take part in neuronal differentiation from immature neuronal precursors to fully differentiated neurons. As a model to study miRNA expression in the developing brain, we prepared cortical brain organoids. The brain organoids comprise rosette structures at early development stages and thus show the different levels of differentiation from precursor neurons to mature neurons. miRNA examples include miR-9, miR-17, and miR-124, and it is discussed how to modify protocol settings for best output.