ABSTRACT
BACKGROUND: Biopsies extracted from brain cancer patients often display degraded ribosomal RNA, which makes them unusable in transcriptomic experiments. This has not been properly documented in previous works aimed at refining the molecular classification of brain cancer. OBJECTIVE: To determine RNA integrity in a large cohort of human brain cancer biopsies and to evaluate different factors that may influence RNA integrity in both a murine model of glioblastoma and in additional subsets of patient biopsies. METHODS: Total RNA was isolated from 255 biopsies of various human brain tumors (HBTs) and processed on a Bioanalyzer. Correct RNA integrity was considered for samples showing either the ribosomal 28S/18S peak ratio ≥ 1.2 or RNA integrity number ≥ 6. The time-dependent effect of ex vivo ischemia was evaluated in a murine model, whose results were tested in a new collection of 27 human biopsies. Multiple biopsy sampling was considered in a further set comprising 32 biopsies. RESULTS: The 255 human biopsies revealed a substantial percentage of samples displaying degraded RNA (27.5%). The murine model confirmed the known relevance of ex vivo ischemia time in increased RNA degradation. Human biopsies extracted immediately after cauterization showed a trend toward less RNA degradation. Combining snap freezing and multiple sampling of biopsies, the percentage of patients with degraded RNA was reduced by twofold (15.6%). CONCLUSIONS: We provide a first concise study of factors influencing RNA degradation in HBT biopsies. Immediate biopsy removal after cauterization of the tumor area, snap freezing, and multiple sampling improve RNA quality.
