Highly efficient and robust π-FISH rainbow for multiplexed in situ detection of diverse biomolecules

Abstract

In the unprecedented single-cell sequencing and spatial multiomics era of biology, fluorescence in situ hybridization (FISH) technologies with higher sensitivity and robustness, especially for detecting short RNAs and other biomolecules, are greatly desired. Here, we develop the robust multiplex π-FISH rainbow method to detect diverse biomolecules (DNA, RNA, proteins, and neurotransmitters) individually or simultaneously with high efficiency. This versatile method is successfully applied to detect gene expression in different species, from microorganisms to plants and animals. Furthermore, we delineate the landscape of diverse neuron subclusters by decoding the spatial distribution of 21 marker genes via only two rounds of hybridization. Significantly, we combine π-FISH rainbow with hybridization chain reaction to develop π-FISH+ technology for short nucleic acid fragments, such as microRNA and prostate cancer anti-androgen therapy-resistant marker ARV7 splicing variant in circulating tumour cells from patients. Our study provides a robust biomolecule in situ detection technology for spatial multiomics investigation and clinical diagnosis.

Single-Cell Liquid Biopsy of Lung Cancer: Ultra-Simplified Efficient Enrichment of Circulating Tumor Cells and Hand-Held Fluorometer Portable Testing

“…The operation required only two reagents and three centrifugation times, taking only 45 min. Compared with other studies (Table S4), the reagents for the pretreatment of the PLACS strategy are simple and easy to obtain, and no advance preparation of materials is required. − Moreover, it is easy to operate, requires no professional personnel, and has a shorter processing time, which is expected to provide a simple and effective way for CTC sample processing.…”

Explore & actuate: the future of personalized medicine in oncology through emerging technologies

“…An actuator technology with great potential is multiplex nanopore sequencing (MNS), allowing bulk-level detection and quantification at multiple-omic levels simultaneously, to about 40 biomarkers [61 ▪▪ ]. π-FISH rainbow is a potent multiomic spatial profiling technology with high sensitivity that can potentially be used to visualize the TME in a targeted manner, while π-FISH+ detects small nucleic acid fragments [62 ▪▪ ]. AmpliFISH is another technology that can help to visualize RNA localization at cellular resolution [63].…”

Designing a Hybrid Chain Reaction Probe for Multiplex Transcripts Assay with High-Level Imaging

“…Tissues consist of diverse cell types, and investigating the spatial organization of these cell types can reveal crucial relationships between cellular state and function within tissues. , Fluorescent in situ hybridization (FISH) is a potent detection method for labeling and imaging nucleic acid molecules, enabling quantitative assays and spatial distribution analysis of nucleic acids within cells or tissues. As a spatial transcriptomic technique, multiplex FISH profiles transcript molecules at the single-cell level, allowing exploration of tissue organization’s molecular signatures. − …”

“…Several FISH imaging methods have been developed, such as single-molecule FISH (smFISH), , branched DNA (bDNA) FISH, , and hybrid chain reaction (HCR). , These methods share the goal of amplifying signals by recruiting multiple fluorescent molecules to a specific RNA molecule. For example, early methods like smFISH employed 10–50 fluorescent molecules tethered to individual mRNA molecules of a target gene, resulting in dim signals due to limited fluorescent molecule binding.…”

“…As a spatial transcriptomic technique, multiplex FISH profiles transcript molecules at the single-cell level, allowing exploration of tissue organization's molecular signatures. 3−6 Several FISH imaging methods have been developed, such as single-molecule FISH (smFISH), 7,8 branched DNA (bDNA) FISH, 5,9 and hybrid chain reaction (HCR). 10,11 These methods share the goal of amplifying signals by recruiting multiple fluorescent molecules to a specific RNA molecule.…”