The spatial RNA integrity number assay for in situ evaluation of transcriptome quality

The RNA integrity number (RIN) is a frequently used quality metric to assess the completeness of rRNA, as a proxy for the corresponding mRNA in a tissue. Current methods operate at bulk resolution and provide a single average estimate for the whole sample. Spatial transcriptomics technologies have emerged and shown their value by placing gene expression into a tissue context, resulting in transcriptional information from all tissue regions. Thus, the ability to estimate RNA quality in situ has become of utmost importance to overcome the limitation with a bulk rRNA measurement. Here we show a new tool, the spatial RNA integrity number (sRIN) assay, to assess the rRNA completeness in a tissue wide manner at cellular resolution. We demonstrate the use of sRIN to identify spatial variation in tissue quality prior to more comprehensive spatial transcriptomics workflows.


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Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Joakim Lundeberg Oct 23, 2020 The present study does not involve data collection. For all spatial transcriptomics (ST) and sRIN tissue experiments: The sample size (number of tissue sections) varied from two to four, which is in the range of most previous published ST experiments. Specimens included both mouse and human, in total we applied the sRIN assay to seven distinct tissue regions. Supplementary Figure 1: Three technical replicates were selected for qPCR using cDNA template originating from total RNA, we expected the variability to be low and considered the selected sample size to be sufficient. For qPCR using cDNA template originating from in situ total RNA, we expected the variaility to be higher and considered the selected sample size of 18 technical replicates to be sufficient. We applied the sRIN assay to different sample types and species, all generating sRIN heat maps showing spatial RNA quality at a single cell resolution. Experimental results demonstrate the sRIN assays ability to measure different cDNA lengths in situ. All attempts at replication were successful.

Supplementary
Supplementary Figure 6: When investigating the sRIN assay's 18S rRNA specificity, when analyzing total RNA samples were divided into separate groups based on the presence or absence of a visible 18S rRNA peak in their respective gel electropherograms.
Blinding was not relevant to this study as there were no stratified comparisons between groups.