INTRODUCTION: To optimize RNA sequencing (RNA-seq) outcomes, we investigated preanalytical variables in malignant effusions containing metastatic breast cancer. We compared 2 processing methods-Ficoll-Hypaque density gradient enrichment and CytoLyt hemolysis-focusing on their effects on RNA quality, transcript abundance, and variant detection from cytospin slides, relative to fresh-frozen samples. Additionally, we compared read-based and Unique Molecular Identifier (UMI)-based library preparation methods. MATERIALS AND METHODS: Thirteen malignant effusion specimens from metastatic breast cancer were processed using both the Ficoll-Hypaque and Cytolyt methods. RNA was extracted from fresh-frozen samples stored in RNA preservative and from cytospin slides fixed in Carnoy's solution. RNA quality was evaluated using RNA integrity number (RIN) and the percentage of fragments >
200 bases (DV200). Sequencing was conducted with both read- and UMI-based methods. RESULTS: Purified RNA was more fragmented by the Cytolyt method (mean RIN: 3.56, DV200: 78.97%), compared to the Ficoll-Hypaque method (mean RIN: 6.29, DV200: 88.08%). Sequencing data had high concordance correlation coefficient (CCC) for measurements of gene expression, whether from Cytolyt or Ficoll-Hypaque treated samples, and whether using the UMI- or read-based sequencing methods (read-based mean CCC: 0.967 from Cytolyt versus 0.974 from Ficoll-Hypaque, UMI-based mean CCC: 0.972 from Cytolyt versus 0.977 from Ficoll-Hypaque). CONCLUSIONS: Despite the increased RNA fragmentation with the Cytolyt, RNA-seq data quality was comparable across Cytolyt and Ficoll-Hypaque methods. Both clearing methods are viable for short-read RNA-seq analysis, with read and UMI-based approaches performing similarly.