Highly Multiplexed Single-Cell In Situ RNA and DNA Analysis by Consecutive Hybridization.

Lu Xiao,Jia Guo,Renjie Liao

doi:10.3390/molecules25214900

Abstract

The ability to comprehensively profile nucleic acids in individual cells in their natural spatial contexts is essential to advance our understanding of biology and medicine. Here, we report a novel method for spatial transcriptomics and genomics analysis. In this method, every nucleic acid molecule is detected as a fluorescent spot at its natural cellular location throughout the cycles of consecutive fluorescence in situ hybridization (C-FISH). In each C-FISH cycle, fluorescent oligonucleotide probes hybridize to the probes applied in the previous cycle, and also introduce the binding sites for the next cycle probes. With reiterative cycles of hybridization, imaging and photobleaching, the identities of the varied nucleic acids are determined by their unique color sequences. To demonstrate the feasibility of this method, we show that transcripts or genomic loci in single cells can be unambiguously quantified with 2 fluorophores and 16 C-FISH cycles or with 3 fluorophores and 9 C-FISH cycles. Without any error correction, the error rates obtained using the raw data are close to zero. These results indicate that C-FISH potentially enables tens of thousands (216 = 65,536 or 39 = 19,683) of different transcripts or genomic loci to be precisely profiled in individual cells in situ.

Highlights

Multiplexed single-cell in situ nucleic acids analysis promises to provide new insights into many fields in biology and medicine, such as neuroscience, cancer biology and precision medicine [1].Next-generation sequencing [2,3] and microarray technologies [4] are powerful tools to profile nucleic acids on a transcriptome- or genome-wide scale
To demonstrate the feasibility of this method, we show that transcripts or genomic loci can be successfully detected with 2 dyes in 16 cycles or with 3 dyes in 9 cycles
In this consecutive fluorescence in situ hybridization (C-fluorescence in situ hybridization (FISH)) approach (Figure 1A), an individual nucleic acid target is first hybridized with a set of pre-decoding probes

Summary

Introduction

Next-generation sequencing [2,3] and microarray technologies [4] are powerful tools to profile nucleic acids on a transcriptome- or genome-wide scale. As these approaches require the nucleic acids to be extracted from the cells and subsequently purified before sequence identification, the cellular location information of the transcripts and genomic loci is lost during analysis. Multicolor karyotyping technologies [5,6,7,8] have been developed to visualize chromosomes in their natural spatial contexts These approaches have not been applied for profiling transcripts or genomic loci. Due to the broad absorption and emission peaks of the common fluorophores, their spectral overlap limits the number of nucleic acids that can be quantified simultaneously in the same specimen

Methods

Results

Conclusion