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In various malignancies RNA fragments are dysregulated. Our study was designed to determine the expression of 4, 5′-tRNA halves in the tissue and serum of patients with clear cell renal cell carcinoma.Tissue and serum samples of patients with clear cell renal cell carcinoma and nonmalignant disease were collected prospectively in our biobank. We isolated total RNA from 95 clear cell renal cell carcinomas and 50 normal renal tissues as well as serum RNA from 27 patients with clear cell renal cell carcinoma and 13 with nonmalignant urological disease. To specifically determine the expression of 5′-tRNA halves we dephosphorylated and ligated an adaptor nucleotide to the 3′ end of the tRNA halves. The expression levels of 4, 5′-tRNA halves (5′-tRNA-Arg-CCT, 5′-tRNA-Glu-CTC, 5′-tRNA-Leu-CAG and 5′-tRNA-Lys-TTT) were then measured by TaqMan® based quantitative reverse transcription-polymerase chain reaction.All studied 5′-tRNA halves were down-regulated in clear cell renal cell carcinoma tissues, indicating a potential role as a tumor suppressor. Furthermore, we noted decreased expression of 5′-tRNA halves in patients with adverse clinicopathological parameters. All 5′-tRNA halves were expressed at lower levels in nonorgan confined clear cell renal cell carcinoma. The 5′-tRNA-Lys-TTT halves inversely correlated with ISUP (International Society of Urological Pathology) grade. In patients with clear cell renal cell carcinoma 5′-tRNA-Arg-CCT, 5′-tRNA-Glu-CTC and 5′-tRNA-Lys-TTT halves circulated at lower levels than in control subjects, indicating relevance as noninvasive biomarkers.In patients with clear cell renal cell carcinoma 5′-tRNA halves have potential as diagnostic and prognostic biomarkers. The 5′-tRNA halves may act in a tumor suppressive manner, which requires further research to confirm.