Assessment of long-term stored specimens in the Siriraj Hospital colorectal cancer biobank for RNA sequencing and profiling

Abstract

Biobanks play an important role in advancing cancer research, yet concerns persist regarding the molecular integrity of long-term stored samples. This study assessed fresh frozen (FF) tissues and formalin-fixed paraffin-embedded (FFPE) tissues from the Siriraj Hospital colorectal cancer (CRC) biobank collected during two distinct periods (2011-2012 and 2020-2021). In 2022, FF and FFPE primary cancer tissues from 75 CRC patients were evaluated. RNA sequencing (RNA-Seq) analyzed comprehensive gene expression profiles in FF tissues preserved at -80 » °C, while nCounter profiling elucidated cancer-specific RNA transcripts in FFPE tissues stored at ambient temperature. Comparative analyses were conducted between specimens from 2011 to 2012 and 2020-2021. The FF tissues stored for approximately 10.5 years were well-suited for RNA-Seq compared to the intact tissues preserved for 1.5 years. Despite consistencies in RNA quantity, RNA integrity, amount of sequencing reads, and CRC gene signature, gene enrichment analysis revealed the decreased ribosome biogenesis, spliceosome and antifolate resistance pathways in the 2011-2012 group. Moreover, the FFPE tissues also showed no alteration in RNA quantity between the two periods, and the nCounter profiling demonstrated comparable CRC-specific gene counts in spite of the significant reduction of raw counts in the 2011-2012 group. We report that FF tissues from CRC patients, stored for 10 years, are viable for whole transcriptome RNA-Seq, despite altered pathways such as ribosome biogenesis, spliceosome, and antifolate resistance. Moreover, 10-year-stored FFPE CRC tissues remain suitable for specific RNA profiling using the nCounter pan-cancer panel, despite a significant reduction in raw counts. These findings underscore the enduring contribution of biobanks to molecular research, highlighting their value a decade post-collection.