Highly specific imaging of mRNA in single cells by target RNA-initiated rolling circle amplification.
Ruijie DengKaixiang ZhangYupeng SunXiaojun RenJinghong LiPublished in: Chemical science (2017)
Detection of single-cell gene expression with high spatial and sequence resolution is a key challenge in single cell biology. Herein, we propose a robust method for the direct detection of mRNA, termed target RNA-initiated rolling circle amplification, which enables imaging of mRNA with single-nucleotide and near-single-molecule resolution in single cells. By utilizing a Splint R ligase capable of efficiently catalyzing the ligation of a padlock probe by the target RNA, the method can enable the efficient detection of mRNA without reverse transcription (detection efficiency over 20%). Meanwhile, attributed to the ligation-based recognition process, the method confers specificity sufficient to genotype mRNAs with one-nucleotide variations. The method has enabled the spatial mapping and correlation analysis of gene expression in single cells which could help us to elucidate the gene functions and regulatory mechanisms. This method offers an mRNA profiling ability with high spatial resolution and sequence specificity, thus is expected to be a single-cell analysis platform for not only investigating gene expression, but also potentially for analyzing single-nucleotide variants or mRNA alternative splicing at single-cell level.
Keyphrases
- single cell
- gene expression
- single molecule
- induced apoptosis
- rna seq
- label free
- high throughput
- cell cycle arrest
- dna methylation
- high resolution
- binding protein
- loop mediated isothermal amplification
- nucleic acid
- real time pcr
- endoplasmic reticulum stress
- signaling pathway
- transcription factor
- cell death
- cell proliferation
- copy number
- genome wide
- living cells
- oxidative stress
- mass spectrometry
- quantum dots
- structural basis
- genome wide analysis