Journal
FRONTIERS IN CELL AND DEVELOPMENTAL BIOLOGY
Volume 6, Issue -, Pages -Publisher
FRONTIERS MEDIA SA
DOI: 10.3389/fcell.2018.00042
Keywords
transcriptomics; genomics; fluorescence in situ hybridization; strand displacement reactions; RNA expression; oligonucleotides; fluorescent probes; single-cell
Categories
Funding
- National Institute of Allergy and Infectious Diseases of the National Institutes of Health [R21AI132840]
- Arizona State University
- Arizona State University/Mayo Clinic seed grant [ARI-219693]
- Cystic Fibrosis Foundation [FIRTH17XX0]
Ask authors/readers for more resources
Comprehensive RNA analyses in individual cells in their native spatial contexts promise to transform our understanding of normal physiology and disease pathogenesis. Here we report a single-cell in situ RNA analysis approach using switchable fluorescent oligonucleotides (SFO). In this method, transcripts are first hybridized by pre-decoding oligonucleotides. These oligonucleotides subsequently recruit SFO to stain their corresponding RNA targets. After fluorescence imaging, all the SFO in the whole specimen are simultaneously removed by DNA strand displacement reactions. Through continuous cycles of target staining, fluorescence imaging, and SFO removal, a large number of different transcripts can be identified by unique fluorophore sequences and visualized at the optical resolution. To demonstrate the feasibility of this approach, we show that the hybridized SFO can be efficiently stripped by strand displacement reactions within 30 min. We also demonstrate that this SFO removal process maintains the integrity of the RNA targets and the pre-decoding oligonucleotides, and keeps them hybridized. Applying this approach, we show that transcripts can be restained in at least eight hybridization cycles with high analysis accuracy, which theoretically would enable the whole transcriptome to be quantified at the single molecule sensitivity in individual cells. This in situ RNA analysis technology will have wide applications in systems biology, molecular diagnosis, and targeted therapies.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available