PMID- 33113917 OWN - NLM STAT- MEDLINE VI - 25 IP - 21 TI - Highly Multiplexed Single-Cell In Situ RNA and DNA Analysis by Consecutive Hybridization. LA - eng PT - Journal Article PL - Switzerland TA - Molecules JT - Molecules (Basel, Switzerland) JID - 100964009 IS - 1420-3049 (Electronic) LID - E4900 [pii] LID - 10.3390/molecules25214900 [doi] FAU - Xiao, Lu AU - Xiao L AD - Biodesign Institute & School of Molecular Sciences, Arizona State University, Tempe, AZ 85287, USA. FAU - Liao, Renjie AU - Liao R AD - Biodesign Institute & School of Molecular Sciences, Arizona State University, Tempe, AZ 85287, USA. FAU - Guo, Jia AU - Guo J AUID- ORCID: 0000-0001-8036-5349 AD - Biodesign Institute & School of Molecular Sciences, Arizona State University, Tempe, AZ 85287, USA. IS - 1420-3049 (Linking) RN - 63231-63-0 (RNA) RN - 9007-49-2 (DNA) SB - IM MH - DNA/*analysis/chemistry MH - HeLa Cells MH - Humans MH - In Situ Hybridization, Fluorescence MH - *Nucleic Acid Hybridization MH - RNA/*analysis/chemistry MH - Single-Cell Analysis/*methods OTO - NOTNLM OT - FISH OT - fluorescence in situ hybridization OT - genomic loci OT - genomics OT - transcriptomics OT - transcripts PMC - PMC7660199 DCOM- 20210331 LR - 20210331 DP - 2020 Oct 23 DEP - 20201023 AB - 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.