Effect of Duration of Ex Vivo Ischemia Time and Storage Period on RNA Quality in Biobanked Human Renal Cell Carcinoma Tissue.

BACKGROUND

RNA degradation is a major problem in tissue banking, and the effects of the ex vivo ischemia time, storage time, and transport conditions on RNA integrity and gene expression have not been well understood.

METHODS

A total of 100 fresh-frozen clear cell carcinoma tissues and matched normal tissues during five storage periods (≤6, 7-12, 13-18, 19-24, and 25-30 months) were chosen to detect RNA quality. At surgery, fresh kidney cancer tissues from five patients were cut into pieces and snap frozen. Additional fresh tissue pieces were (1) left at room temperature, (2) kept on ice, or (3) placed in normal saline before being snap frozen after 0.5, 1, 2, or 4 h. RNA integrity was determined by microchip electrophoresis, and gene expression was analyzed by real-time polymerase chain reaction.

RESULTS

Altogether, 82 % of kidney cancer specimens banked using a standardized protocol yielded RNA with an RNA integrity number of ≥7 (7.77 ± 0.95, 7.87 ± 0.37, 7.15 ± 1.46, 8.10 ± 0.64, and 7.11 ± 1.08 during the five storage periods, respectively). RNA remained intact after 4 h on ice, whereas degradation was found in tissues left at room temperature or in saline. Expression of genes in certain functional pathways changed during storage under three conditions.

CONCLUSIONS

More than 80 % of the banked kidney cancer biospecimens collected following a standardized protocol yielded high-quality RNA. Fresh human cancer tissue samples should be transported on ice before biobanking to avoid a major reduction in RNA quality. The presented data should be considered in attempts to further standardize tissue biospecimen collection and banking.