Evaluating transcriptome-wide information preservation by ensilication using long-read RNA sequencing

Abstract

Background. Evaluation of RNA preservation requires an understanding of how storage conditions affect recoverable transcriptome information. Long-read RNA sequencing enables comprehensive assessment of transcript representation, isoform composition, and support for annotated transcript structures. This approach was employed to investigate the protective capabilities of ensilication during ambient and elevated-temperature storage. Results. Pooled human total RNA was analyzed using PacBio Kinnex across six conditions, with two replicates per storage condition. Comparisons included frozen controls as well as ensilicated and unprotected RNA stored for seven days at room temperature or 40 {degrees}C. At matched input depth, with at least one qualifying sequence per transcript, ensilicated samples retained 88-89% of a fixed frozen-reference transcript set at room temperature and approximately 85% at 40 {degrees}C. In contrast, unprotected RNA retained 78-83% and 42-64%, respectively. Ensilicated samples exhibited smaller changes in isoform composition among evaluable genes. The retention advantage persisted when sequences were required to support a uniquely distinguishing complete intron chain and both annotated transcript boundaries. Retention estimates varied with detection criteria, and substantial limitations in annotated end-to-end coverage were observed even in frozen controls. Conclusion. Long-read RNA sequencing demonstrates that ensilication limits storage-associated loss and distortion of recoverable transcriptome-wide information in the tested RNA preparation. These findings indicate a protective effect during seven days of ambient and elevated-temperature storage.