Highly multiplexed single-cell in situ RNA and DNA analysis with bioorthogonal cleavable fluorescent oligonucleotides.

ABSTRACT: The ability to profile transcripts and genomic loci comprehensively in single cells in situ is essential to advance our understanding of normal physiology and disease pathogenesis. Here we report a highly multiplexed single-cell in situ RNA and DNA analysis approach using bioorthogonal cleavable fluorescent oligonucleotides. In this approach, oligonucleotides tethered to fluorophores through an azide-based cleavable linker are used to detect their nucleic acids targets by in situ hybridization. After fluorescence imaging, the fluorophores in the whole specimen are efficiently cleaved in 30 minutes without loss of RNA or DNA integrity. Through reiterative cycles of hybridization, imaging, and cleavage, this method has the potential to quantify hundreds to thousands of different RNA species or genomic loci in single cells in situ at the single-molecule sensitivity. Applying this approach, we demonstrate that different nucleic acids can be detected in each hybridization cycle by multi-color staining, and at least ten continuous hybridization cycles can be carried out in the same specimen. We also show that the integrated single-cell in situ analysis of DNA, RNA and protein can be achieved using cleavable fluorescent oligonucleotides combined with cleavable fluorescent antibodies. This highly multiplexed imaging platform will have wide applications in systems biology and biomedical research.

SUBMITTER: Mondal M 

PROVIDER: S-EPMC5914540 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature