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Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue
Published on: September 2, 2013
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Transcriptomic profiling of nuclei from paraformaldehyde-fixed and formalin-fixed paraffin-embedded brain tissues
Yunxia Guo1, Junjie Ma2, Zhengyue Li1
1State Key Laboratory of Digital Medical Engineering, School of Biological Science & Medical Engineering, Southeast University, Nanjing, 210096, China.
Analytica Chimica Acta
|May 24, 2024
Summary
Paraformaldehyde (PFA) fixation duration significantly impacts brain nuclear transcriptome profiles, altering cell states and gene expression. FFPE storage time has minimal effect, guiding single-nucleus RNA-seq preparation for brain disease studies.
Area of Science:
- Neuroscience
- Genomics
- Pathology
Background:
- Formalin-fixed and paraffin-embedded (FFPE) tissues are crucial for histochemical staining and clinical research.
- Single-cell gene expression analysis is emerging for FFPE specimens, with single-nuclear transcriptomics beneficial for challenging tissues like the brain.
- The impact of standard pathological fixation on archived FFPE sample transcriptomes is not well-understood.
Purpose of the Study:
- To investigate the effects of paraformaldehyde (PFA) fixation and paraffin-embedding on the nuclear transcriptome of mouse hippocampus.
- To assess the influence of fixation duration and FFPE storage time on transcriptional profiles and cell composition.
Main Methods:
- RNA sequencing (RNA-seq) was performed on nuclei isolated from fresh PFA-fixed, FFPE, and cryopreserved mouse hippocampus samples.
- Transcriptomic profiles and cell type proportions were analyzed in relation to fixation duration (1-day vs. 3-day PFA) and FFPE storage time.
Main Results:
- Transcriptomic signatures of fresh PFA-fixed and FFPE tissues were comparable to cryopreserved samples.
- Extended PFA fixation (>3 days) increased differentially expressed genes, particularly in spliceosome and synaptic pathways.
- PFA fixation altered cell states, decreasing neuronal and oligodendrocyte populations while increasing microglia; FFPE storage time had minimal impact on cell composition.
Conclusions:
- Fixation time, especially between 1-3 days, significantly influences nuclear transcriptomic profiles and cell states in brain tissue more than FFPE storage duration.
- These findings provide critical guidance for optimizing sample preparation in single-nucleus RNA-seq studies of FFPE brain samples for disease research.
