Transcriptomic profiling of nuclei from paraformaldehyde-fixed and formalin-fixed paraffin-embedded brain tissues.
Anal Chim Acta
; 1281: 341861, 2023 Nov 15.
Article
em En
| MEDLINE
| ID: mdl-38783731
ABSTRACT
BACKGROUND:
Paraformaldehyde (PFA) fixation is necessary for histochemical staining, and formalin-fixed and paraffin-embedded (FFPE) tissue archives are the largest repository of clinically annotated specimens. Single-cell gene expression workflows have recently been developed for PFA-fixed and FFPE tissue specimens. However, for tissues where intact cells are hard to recover, including tissues containing highly interconnected neurons, single-nuclear transcriptomics is beneficial. Moreover, since RNA is very unstable, the effects of standard pathological practice on the transcriptome of samples obtained from such archived specimens like FFPE samples are largely anecdotal.RESULTS:
We evaluated the effects of polyformaldehyde (PFA) fixation and paraffin-embedding on transcriptional profiles of the mouse hippocampus obtained by RNA sequencing (RNA-seq). The transcriptomic signatures of nuclei isolated from fresh PFA-fixed and fresh FFPE tissues were comparable to those of cryopreserved samples. However, more differentially expressed genes were obtained for brains after PFA fixation for more than 3 days than in fresh PFA-fixed samples, especially genes involved in spliceosome and synaptic-related pathways. Importantly, the real cell states were destroyed, with oligodendrocyte precursor cells depleted in the 1day fixed hippocampus. After fixation for 3 days, the proportions of neuronal cells and oligodendrocytes decreased and microglia increased; however, relative frequencies remained constant for longer fixation durations. The storage time of FFPE samples had a negligible effect on the cell composition.SIGNIFICANCE:
This represents the first work to investigate the effects of fixation and storage time of brains on its nuclear transcriptome signatures in detail. The fixation time had more influences on the nuclear transcriptomic profiles than FFPE retention time, and the cliff-like effects appeared to occur over a fixed period of 1-3 days. These findings are expected to guide sample preparation for single-nucleus RNA-seq of FFPE samples, particularly in transcriptomic studies focused on brain diseases.Palavras-chave
Texto completo:
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Fixação de Tecidos
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Inclusão em Parafina
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Perfilação da Expressão Gênica
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Formaldeído
Limite:
Animals
Idioma:
En
Revista:
Anal Chim Acta
Ano de publicação:
2023
Tipo de documento:
Article