High-Resolution RNA Sequencing from PFA-Fixed Microscopy Sections.
Microdissection
PFA-fixation
RNA extraction
RNA sequencing
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:
2023
2023
Historique:
entrez:
30
1
2023
pubmed:
31
1
2023
medline:
2
2
2023
Statut:
ppublish
Résumé
Obtaining high-quality RNA sequencing results from archived biological tissues, such as paraformaldehyde (PFA)-fixed sections for microscopy, is challenging due to the incompatibility of current high-throughput RNA sequencing methods. Here, we present a low-input method for RNA sequencing from archived PFA-fixed sections. Using this method, we routinely obtain high-quality sequencing results from archived mouse brain sections that are prepared for imaging without any special care for avoiding RNA degradation. The PFA cross-linking locks and protects RNA from degradation but cross-linking is also hard to reverse. For this goal, we developed an effective decrosslinking protocol based on Proteinase K activity to retrieve PFA-cross-linked mRNAs which was followed up by a Smart-seq2 library preparation protocol. Our protocol enables spatially defined transcriptomic analysis of archived sections and allows the genomic analysis of PFA-fixed samples. Furthermore, our protocol inactivates pathogenic samples and allows working under regular biosafety levels.
Identifiants
pubmed: 36715937
doi: 10.1007/978-1-0716-2926-0_16
doi:
Substances chimiques
RNA
63231-63-0
RNA, Messenger
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
205-212Informations de copyright
© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
Références
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doi: 10.1038/s41467-021-25871-2
Picelli S, Bjorklund AK, Faridani OR et al (2013) Smart-seq2 for sensitive full-length transcriptome profiling in single cells. Nat Methods 10:1096–1098
doi: 10.1038/nmeth.2639
Safaiyan S, Besson-Girard S, Kaya T et al (2021) White matter aging drives microglial diversity. Neuron 109:1100–1117 e1110
doi: 10.1016/j.neuron.2021.01.027
Thomsen ER, Mich JK, Yao Z et al (2016) Fixed single-cell transcriptomic characterization of human radial glial diversity. Nat Methods 13:87–93
doi: 10.1038/nmeth.3629