RNAscope™ In Situ Hybridization for the Detection of a Long Noncoding RNA in an Organ-on-a-Chip.

RNAscope™ is a highly sensitive technique for detecting RNA molecules within cells, offering superior specificity and signal amplification compared with traditional in situ hybridization methods. This technique enables the visualization of gene expression patterns at the single-cell level, making it particularly advantageous for studies involving complex tissues or 3D culture systems. In this study, RNAscope™ was used to investigate gene expression within an organ-on-a-chip (OoC), a system that usually encompasses a limited number of cells making the application of standard gene expression analysis techniques, such as qPCR, difficult. In this study, human articular chondrocytes (Chs) isolated from the knee joints of both osteoarthritic patients and heathy donors' cells were encapsulated in a hyaluronic acid (HA)-based hydrogels and injected in a three-channels microfluidic device. RNAscope™ technique was employed directly within the OoC device to study the expression of a long noncoding transcript, FOXCUT, together with FOXC1 and IL-6 in healthy and diseased chondrocytes and there was a correlation between the methylation profile of FOXCUT and osteoarthritis (OA). RNAscope™ enabled the visualization of the spatial localization of target transcripts, including those with low expression levels, such as FOXC1 and FOXCUT. The standard protocol, originally designed for fixed-frozen tissue samples, required optimization to accommodate the unique properties of the OoC environment. Key adaptations included the fixation step using paraformaldehyde, permeabilization with Triton X-100 and an adjusted protease treatment. These modifications were essential to ensure efficient probe penetration and accurate signal detection within the HA 3D matrix. A detailed protocol is herein provided for the detection of RNA within a microfluidic device by using the RNAscope™ technology.