Chronic opioid use in men results in changes in the ratio of tRF-Gly isoforms in sperm. Quantification of tRF using digital-PCR. Mature sperm cells were purified by a 45–90% Percoll gradient and small RNA was isolated using a Nucleospin kit. cDNA was generated using one specific stem–loop RT primer that binds to the ‘long’ tsRNA isoform (53 nucleotides long) or another specific RT-primer that targets the ‘short’ tsRNA variant (32 nucleotides long). Digital PCR was performed using a tRF-Gly-specific forward primer, a universal reverse primer on the stem loop adapter, and internal Taqman probes targeting either the short or long tsRNA. Results are expressed as: (A) The concentration of ‘long’ tRF Gly-GCC isoforms per microliter of cDNA. (B) The concentration of ‘short’ tRF Gly-GCC isoforms per microliter of cDNA. (C) The ratio of ‘long’ versus ‘short’ tRF Gly-GCC isoforms. (D) The concentration of miR100-5p per microliter of cDNA. ‘C’: Controls (men who do not inject drugs); ‘PWID’: people who inject drugs. P-value was calculated by one-tailed Mann–Whitney U-test. Product specificity was validated by gel-electrophoresis and by absence of products in non-template and non-RT controls. Control group: n = 10, PWID group: included = 13.