HT-smFISH: a cost-effective and flexible workflow for high-throughput single-molecule RNA imaging.
Journal
Nature protocols
ISSN: 1750-2799
Titre abrégé: Nat Protoc
Pays: England
ID NLM: 101284307
Informations de publication
Date de publication:
01 2023
01 2023
Historique:
received:
08
11
2021
accepted:
04
07
2022
pubmed:
26
10
2022
medline:
14
1
2023
entrez:
25
10
2022
Statut:
ppublish
Résumé
The ability to visualize RNA in its native subcellular environment by using single-molecule fluorescence in situ hybridization (smFISH) has reshaped our understanding of gene expression and cellular functions. A major hindrance of smFISH is the difficulty to perform systematic experiments in medium- or high-throughput formats, principally because of the high cost of generating the individual fluorescent probe sets. Here, we present high-throughput smFISH (HT-smFISH), a simple and cost-efficient method for imaging hundreds to thousands of single endogenous RNA molecules in 96-well plates. HT-smFISH uses RNA probes transcribed in vitro from a large pool of unlabeled oligonucleotides. This allows the generation of individual probes for many RNA species, replacing commercial DNA probe sets. HT-smFISH thus reduces costs per targeted RNA compared with many smFISH methods and is easily scalable and flexible in design. We provide a protocol that combines oligo pool design, probe set generation, optimized hybridization conditions and guidelines for image acquisition and analysis. The pipeline requires knowledge of standard molecular biology tools, cell culture and fluorescence microscopy. It is achievable in ~20 d. In brief, HT-smFISH is tailored for medium- to high-throughput screens that image RNAs at single-molecule sensitivity.
Identifiants
pubmed: 36280749
doi: 10.1038/s41596-022-00750-2
pii: 10.1038/s41596-022-00750-2
doi:
Substances chimiques
RNA
63231-63-0
Types de publication
Journal Article
Review
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
157-187Informations de copyright
© 2022. Springer Nature Limited.
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