Getting the message: low-plex detection of RNA in tissues

The rapidly emerging field of spatial transcriptomics is transforming developmental biology, oncology, and disease pathology, mapping intricate gene expression patterns within their native tissue contexts. Traditionally, the analysis of messenger RNA (mRNA) expression has been the main focus of this research. However, the eukaryotic cell transcriptome comprises a diverse array of dynamic RNA molecules. Therefore, spatial profiling of different RNA species throughout their life cycle is essential for comprehensively elucidating RNA biology in complex tissues. Formalin-fixed paraffin-embedded (FFPE) samples are widely used in research and clinical settings optimally preserving tissue morphology and cellular details over long periods. Pathology departments have accrued vast collections of FFPE blocks, creating a rich yet under-utilized array of materials that, accompanied by clinical data, stands as a treasure trove for human biology and translational research. Nevertheless, FFPE specimens present several challenges. The RNA within these samples is prone to fragmentation during processing and may further degrade if stored improperly. Additionally, chemical modifications can occur in the RNA, leading to fragmentation or resistance to enzymatic reactions essential for sequencing. The loss of poly-A tails adds another layer of complexity, limiting the effectiveness of oligo-dT primed reverse transcription. As a result, options for spatial profiling of RNA molecules in this tissue type are restricted. Commercial assays that are now available for both manual and automated detection of single to low-plex mRNA targets in tissues, including point mutations, miRNAs and even oligonucleotides have brought these applications into more mainstream workflows, and these will be the primary focus of this review.