In situ quality control assay generates a spatial heat map of RNA integrity with single cell resolution
Kvastad L1, Carlberg K1, Magnusson J2, Lundeberg J1*.
1Science for Life Laboratory, Division of Gene Technology, KTH Royal Institute of Technology, SE-106 91 Stockholm, Sweden; 2Department of Cell and Molecular Biology, Karolinska Institute, SE-171 77 Stockholm, Sweden
Single cell transcriptomics is a rapidly evolving field. Combined with new technology that includes spatial information, large data sets with high resolution can be obtained to study heterogeneity within a tissue. Also, when preparing samples for sequencing, it is important to determine the quality of the RNA. Thus, we asked whether it would be possible to obtain, in one assay; spatial RNA integrity information, staining for cell morphology enabling pathological analysis, compatibility with both human and mouse tissue as well as compatibility with both crosslinking and non-crosslinking fixatives at a single cell resolution - using only a single tissue section. Building on the recently described Spatial Transcriptomics method  our results show that it is indeed possible. By placing a single cell layer of tissue onto a glass slide, pre-printed with a DNA oligo for rRNA capture, we perform fixation, followed by hematoxylin and eosin staining for imaging, after which we perform overnight cDNA synthesis. Tissue and rRNA template is later removed and fluorescently labeled probes are sequentially hybridized at multiple sites into the cDNA. Here we present an assay capable of investigating spatial RNA integrity in tissue at a single cell level. The results suggest a useful quality control assay for spatial transcriptomics RNA sequencing data and to facilitate evaluation of archival FFPE samples from tissue banks.
Reference:  Ståhl PL and Salmén F et al Visualization and analysis of gene expression in tissue sections by spatial transcriptomics. Science. 2016, 353(6294):78-82
Acknowledgments: work supported by funds from the Swedish Childhood Cancer Foundation (Barncancerfonden).