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Transcriptome Sequencing Across Tissue Storage Conditions

Comparing Gene Expression in Fresh, Frozen, and Fixed Tissues


A recent paper by Jacobsen et al (2023) presents data comparing whole transcriptome sequencing from fresh, frozen and formalin-fixed paraffin-embedded (FFPE) cardiac tissue. This is to investigate the effect of each storage condition on gene expression profiles. The authors investigated the alignment of whole transcriptome sequencing (WTS) data to assess the proportion of off-target reads and abundance of various RNA types and compared the global gene expression patterns. In addition, the concordance of RNA and DNA genotypes for variants of cardiac-disease relevant genes was compared.

Analyzing Gene Expression and Variation in Different Storage Conditions

  • RNA quality was evaluated for each storage condition. As expected, RNA isolated from FFPE tissue was of lower quality compared to RNA from fresh and frozen tissue.
  • Fresh and frozen tissues alignment were similar and consistent among patients, with a large proportion of reads corresponded to active regions of the genome. FFPE samples yielded many reads that were too short to align, inter-individual differences and slightly lower GC content in the aligned reads. The percentage of gene-annotated reads from protein-coding genes were: fresh: 76%, frozen: 73% and FFPE: 48%.
  • Multidimensional scaling (MDS) plots, generated to assess the protein-coding RNA, mtRNA and lncRNA, showed that the variation induced by storage conditions seemed larger than the biological differences among individuals.
  • Spearman’s correlation analysis was performed to assess the intra-individual ranked gene (ranked by log2(CPM)). A very high correlation was found for all three storage conditions when comparing protein coding transcripts and mitochondrial subset between fresh and frozen tissue gene expression profiles. The FFPE tissue’s subset of mitochondrial transcripts seemed falsely overexpressed. Most of the variation between storage conditions for protein-coding and lncRNA subsets was observed in the more weakly expressed genes.
  • Thresholding to filter out genes with low expression levels showed a reduction of the overall variation caused by storage conditions. The protein-coding subset revealed separation of patients and interindividual differences were observed.
  • Concordance between RNA and DNA variants in exon regions of 31 cardiac genes were investigated by comparing data from WST and OMNI5-4 (SNP) array. A high concordance between DNA and RNA variants were found across all storage conditions. FFPE tissue had a statistically significantly higher number of discordant variant calls and larger proportion of variants with insufficient read depths (<75).



In summary, the authors found that fresh, frozen and FFPE tissues can be used to generate reliable gene expression when implementing proper quality control steps. Understanding the pitfalls, and hence possible limitations, of using archival tissue is crucial given the vast historic resource of banked frozen and FFPE samples available to assist medical research and drug development.


Epistem Services

Similar to the techniques outlined in this paper, Epistem regularly employs qPCR and NGS services for gene expression, metagenomic, whole-exome, genotyping and epigenetic analysis from fresh, frozen and FFPE tissue.

Laser Capture Microscopy (LCM) is a powerful tool used at Epistem to select and enrich a population of individual cells within a tissue for input into downstream transcriptomic analysis.

The Histology and Immunohistochemistry team at Epistem have a wealth of experience in developing IHC protocols for a range of biological targets. Our deep understanding of IHC methodology has allowed us to develop and troubleshoot hundreds of working protocols for both automated (Ventana Discovery Ultra) and manual applications. We offer a bespoke service which could be suited to your project. We can work readily with FFPE, frozen tissues, with chromogenic and fluorescent detection systems as well as RNAScope methodologies.