Quantitative Assessment of Subcellular RNA Loss in FFPE Spatial Transcriptomics Restricted; Files Only

Abstract

Spatial transcriptomics technologies are rapidly advancing toward subcellular resolution, enabling direct measurement of RNA distribution within individual cells. However, how tissue preservation affects subcellular RNA organization remains unclear. Here, we systematically characterize RNA loss in formalin-fixed para”n-embedded (FFPE) samples relative to fresh-frozen (FF) tissue at subcellular resolution. Using publicly available spatial transcriptomics datasets, we analyze gene-level transcript counts and quantify RNA distribution across cellular compartments. We find that RNA loss in FFPE is imbalanced between nuclear and cytoplasmic compartments and varies across genes. These patterns are consistent across cell types and tissues, indicating a systematic and gene-dependent distortion of subcellular RNA distribution. Overall, our work identifies a previously unrecognized source of bias in FFPE spatial transcriptomics data. This provides a quantitative framework for its characterization, with implications for the analysis and interpretation of high-resolution spatial transcriptomics datasets.

Table of Contents

1 Introduction 1

2 Results 2

2.1 Overview of analytical framework 2

2.2 Comparison of aggregated transcript counts for matched FF and FFPE samples 2

2.3 Extranuclear RNA loss is independent of cell type 3

2.4 Shared genes exhibit recurrent extranuclear RNA loss patterns 9

2.5 Extranuclear RNA loss is consistent across diverse FFPE cancer samples 9

2.6 Gene structural features are associated with differential extranuclear RNA loss 11

3 Methods 13

3.1 Gene-level analysis of aggregated transcript counts 13

3.2 Cell type clustering and annotation 13

3.3 Cell type–specific extranuclear ratio analysis 14

3.4 Shared gene consistency analysis 14

3.5 Gene structural feature analysis 16

4 Discussion 16

About this Master's Thesis

Rights statement

• Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.

School	Rollins School of Public Health
Department	Biostatistics
Subfield / Discipline	Biostatistics - MPH & MSPH
Degree	M.S.
Submission	Master's Thesis
Language	English
Research Field	Biology, Genetics Biology, Bioinformatics Biology, Biostatistics
Keyword	FFPE Spatial Transcriptomics Subcellular RNA Loss
Committee Chair / Thesis Advisor	Hu, Jian, Emory University
Committee Members	Yang, Jingjing, Emory University

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