Abstract
Small RNAs are non-coding RNAs that play important roles in the life of both animals and plants. They are 21-to 24-nt in length and around 10 nm in size. Their small size and high diversity has made it challenging to develop methods that have sufficient resolution and specificity to multiplex and quantify. We created a method for the detection of small RNA with nanometer resolution by combining the super-resolution method, DNA-based points accumulation in nanoscale topography (DNA-PAINT), and the specificity of locked nucleic acid (LNA) probes for the in situ detection of multiple small RNAs. The method relies on designing probes to target small RNAs that combine the DNA oligos for PAINT with the LNA-containing oligos for hybridization; therefore, we developed an online tool called “Vetting & Analysis of RNA for in situ Hybridization probes” (VARNISH) for probe design. Our method utilizes advances in DNA-PAINT methodologies, including qPAINT for quantification and Exchange-PAINT for multiplexing. We demonstrated these capabilities or sRNA-PAINT by detecting and quantifying small RNAs in different cell layers of early developmental stage maize anther that are important for male sexual reproduction.
