Determining Long Noncoding RNA Mechanisms in Stem Cells using Automated Image Analysis and Single Molecule RNA-Fluorescent in Situ Hybridization

Long noncoding RNAs (lncRNAs) are increasingly recognized as involved in human physiology and disease. Antisense lncRNAs ubiquitous the genome have many mechanisms of action proposed, but single-cell single-molecule information that is needed to support or refute these lacking. Although single molecule RNA-Fluorescent Situ Hybridization (RNA-FISH) a method sometimes used investigate lncRNAs, counts spatial localization often ignored. One reason lack easily implemented broadly applicable experimental computational approaches for utilizing this data. We propose an RNA-FISH-based quantitative approach test different gene regulation by antisense transcription. applied our mammalian lncRNA pair Tsix Xist known inhibitory interactions. two-color, RNA-FISH image acquisition analysis pipelines, we were able quantify at resolution more than 8,000 cells including several biological replicas. Our revealed there was no significant colocalization molecules, providing evidence against inhibition involving binding two transcripts. also found anticorrelation between only occurred sites transcription, which suggests cis based on act Surprisingly, data intronic probes provided transcriptional interference playing role. Colocalization transcription with histone marks elucidated possible relationships active chromatin state. This demonstrates importance when investigating RNA generalizable pipeline can be transcripts across diverse species, cell types, imaging modalities.