In Situ Sequencing: A High-Throughput, Multi-Targeted Gene Expression Profiling Technique for Cell Typing in Tissue Sections.
In situ sequencing
Padlock probe
Rolling circle amplification
Single-cell
Single-molecule
Spatial transcriptomics
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2020
2020
Historique:
entrez:
13
5
2020
pubmed:
13
5
2020
medline:
10
3
2021
Statut:
ppublish
Résumé
Recent advances of image-based in situ mRNA quantification methods allow to visualize where in a tissue section a set of genes is expressed. It enables to map large numbers of genes in parallel and by capturing cellular boundaries allows to assign genes to cells. Here, we present a high-throughput, multi-targeted gene expression profiling technique called in situ sequencing that is capable of localizing hundreds of genes simultaneously and supports cell type classifications that follow transcriptome-based taxonomy. In situ sequencing is a targeted, amplified, and barcoded approach using padlock probes (PLPs) and rolling circle amplification (RCA). The current protocol relies on mRNA fixation, mRNA reverse transcription, residual mRNA degradation, and PLP hybridization. PLPs are amplified by RCA and labeled with fluorophore-conjugated probes, allowing their detection under conventional fluorescence microscopes.
Identifiants
pubmed: 32394391
doi: 10.1007/978-1-0716-0623-0_20
doi:
Substances chimiques
RNA, Messenger
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
313-329Références
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