miRNA in situ hybridization in formaldehyde and EDC–fixed tissues

Conventional in situ hybridization protocols lead to loss of microRNAs, which diffuse out of the formaldehyde-fixed sample owing to their small size. Adding a carbodiimide that stably links the microRNA with the protein matrix around it prevents this diffusion and allows detection of miRNAs at very low expression levels. MicroRNAs are small regulatory RNAs with many biological functions and disease associations. We showed that in situ hybridization (ISH) using conventional formaldehyde fixation results in substantial microRNA loss from mouse tissue sections, which can be prevented by fixation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide that irreversibly immobilizes the microRNA at its 5′ phosphate. We determined optimal hybridization parameters for 130 locked nucleic acid probes by recording nucleic acid melting temperature during ISH.