Quantitative RNA assessment and long-term stability in the FFPE tumor samples using Digital Spatial Profiler.
Author(s): Gupta S, Chen T, Destenaves B
Publication: Immunooncol Technol, 2022, Vol. 13, Page 100069
PubMed ID: 35754852 PubMed Review Paper? No
Purpose of Paper
This paper investigated the impact of storing paraffin-dipped and undipped slides at 4°C for different durations on RNA counts using a Digital Spatial Profiler. Two archival formalin-fixed paraffin embedded (FFPE) bladder tumor specimens were used for analysis.
Conclusion of Paper
Hierarchical clustering based on RNA counts separated section regions containing tumor (CK+) from those in the tumor microenvironment (CD45+) but did not separate based on whether or not slides were dipped in paraffin or the duration of 4°C storage. RNA expression was very strongly correlated between storage timepoints and between undipped and paraffin-dipped slides at each storage timepoint. While the percentage of RNAs with sufficient signal did not differ between undipped and paraffin-dipped slides at any of the storage timepoints evaluated, the number of RNAs with sufficient signal was more consistent during storage when the slides were dipped in paraffin prior to storage. None of the individual RNAs showed consistent loss of signal with longer storage, regardless of whether they were coated in paraffin.
Studies
-
Study Purpose
This study investigated the impact of storing paraffin-dipped and undipped FFPE slides at 4°C for different durations on RNA counts using a Digital Spatial Profiler. FFPE bladder cancer specimens from two male patients (age 53 and 62) with high grade carcinomas were acquired commercially (processing details not provided). The FFPE blocks were stored for 5 and 8 years, before sectioning. One section from each block was H&E stained to confirm the inclusion of tumor regions. Four sections (3, 5, 7 and 11) from each block were dipped in paraffin, while the other four (2, 4, 6 and 10) were not. Slides were stored at 4°C in a low humidity environment (with a desiccator). At each storage timepoint (0, 16, 24 and 32 weeks) a pair of consecutive slides of each type (paraffin-dipped, not-dipped) from each block were used for RNA digital spatial profiling. Spatial profiling of 84 RNAs was conducted using the GeoMx Immune Pathway Panel human RNA core and antibodies against cytokeratin and CD45 and a GeoMx DSP instrument. Similar regions of interest that were CK positive (tumor) and CD45 positive (tumor microenvironment) were analyzed.
Summary of Findings:
Hierarchical clustering based on RNA counts separated regions containing tumor (CK+) from those in the tumor microenvironment (CD45+) but did not separate whether or not slides were dipped in paraffin or the duration of 4°C storage. RNA expression was very strongly correlated between storage timepoints both in undipped (R=0.96-0.97) and paraffin-dipped (R=0.96-0.97) slides. RNA expression was also strongly correlated between undipped and paraffin-dipped slides at each storage timepoint (R>0.97, all). The percentage of RNA with sufficient signal did not differ between undipped and paraffin-dipped slides at any of the storage timepoints, but the number of RNAs with sufficient signal was more consistent during storage when slides were dipped in paraffin. None of the individual RNAs evaluated showed consistent loss of signal with longer storage, regardless of whether slides were paraffin-dipped.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform RNA In situ hybridization Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage conditions Paraffin-dipped slide
Undipped slide
Storage Storage duration 0 weeks
16 weeks
24 weeks
36 weeks