Comparison of whole transcriptome sequencing of fresh, frozen, and FFPE cardiac tissue

The use of fresh tissue for molecular studies is preferred but often impossible. Instead, frozen or formalin-fixed, paraffin-embedded (FFPE) tissues are widely used and constitute valuable resources for retrospective studies. Researchers at the University of Copenhagen assessed the utility of cardiac tissue stored in different ways for gene expression analyses by whole transcriptome sequencing of paired fresh, frozen, and FFPE tissues. RNA extracted from FFPE was highly degraded. Sequencing of RNA from FFPE tissues yielded higher proportions of intronic and intergenic reads compared to RNA from fresh and frozen tissues. The global gene expression profiles varied with the storage conditions, particularly mitochondrial and long non-coding RNAs. However, the researchers observed high correlations among protein-coding transcripts (ρ > 0.94) with the various storage conditions. They did not observe any significant storage effect on the allele-specific gene expression. However, FFPE had statistically significantly (p < 0.05) more discordant variant calls compared to fresh and frozen tissue.

Graphical representation of the workflow

*Only one RNA extraction from each storage condition from patient 5. Abbreviations: DV200: Percentage of RNA fragments > 200 nucleotides, FFPE: Formalin-fixed, paraffin-embedded, RIN: RNA integrity number, SNP = Single nucleotide polymorphism.

In conclusion, the researchers found that frozen and FFPE tissues can be used for reliable gene expression analyses, provided that proper quality control is performed and caution regarding the technical variability is withheld.

Jacobsen SB, Tfelt-Hansen J, Smerup MH, Andersen JD, Morling N (2023) Comparison of whole transcriptome sequencing of fresh, frozen, and formalin-fixed, paraffin-embedded cardiac tissue. PLoS ONE 18(3): e0283159. [article]

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