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Updated: Aug 12, 2025
Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue
Published on: August 6, 2009
High-Resolution RNA Sequencing from PFA-Fixed Microscopy Sections
Hao Ji1, Simon Besson-Girard1, Peter Androvic1
1Systems Neuroscience Laboratory, Institute for Stroke and Dementia Research (ISD), Klinikum der Universität München, Munich, Germany.
Abstract:
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.
Insights
Researchers developed a new method for RNA sequencing from archived, paraformaldehyde-fixed (PFA) tissue sections. This technique retrieves cross-linked mRNA, enabling high-quality transcriptomic analysis of valuable historical samples.
Area of Science:
- Molecular Biology
- Genomics
- Histology
Background:
- Archived biological tissues, particularly paraformaldehyde (PFA)-fixed sections used in microscopy, present challenges for current RNA sequencing (RNA-seq) methods.
- PFA fixation cross-links and protects RNA but also hinders its retrieval, limiting transcriptomic analysis of historical samples.
Purpose of the Study:
- To develop a low-input RNA sequencing method compatible with archived PFA-fixed tissue sections.
- To enable high-quality transcriptomic analysis and genomic analysis of PFA-fixed samples.
Main Methods:
- A novel decrosslinking protocol utilizing Proteinase K activity was developed to retrieve PFA-cross-linked messenger RNAs (mRNAs).
- The retrieved mRNAs were then processed using the Smart-seq2 library preparation protocol.
- The method was validated using archived mouse brain sections prepared for imaging.
Main Results:
- The developed protocol successfully yielded high-quality RNA sequencing results from archived PFA-fixed mouse brain sections.
- The method allows for spatially defined transcriptomic analysis of archived sections.
- The protocol inactivates pathogenic samples, enabling work under regular biosafety levels.
Conclusions:
- This low-input RNA sequencing method effectively retrieves and analyzes RNA from archived PFA-fixed sections.
- The protocol expands the utility of historical tissue samples for genomic and transcriptomic studies.
- The method offers a safe and accessible approach for analyzing PFA-fixed specimens.
