To quantify the level of 185 prevalent tRFs or tRNA halves (tiRNAs) in sperm cells from PWID and controls, we used the nrStart™ tRF&tiRNA PCR array (catalog: AS-NR-002-1, ArrayStar, Rockville, MD, USA). The array covers all the nuclear anticodon isoacceptors catalogued in the genomic tRNA database (GtRNAdb), providing insights on both the isoacceptor and the specific isodecoder expression. The total RNA purified from sperm cells as described above was submitted to ArrayStar PCR service. An rtStar™ tRF&tiRNA Pretreatment Kit (Cat# AS-FS-005, Arraystar, Rockville, MD, USA) was used for RNA sample pretreatment; this process removed chemical modifications such as methylation and acylation from tRF and tiRNA. After phenol–chloroform extraction and ethanol precipitation, rtStar™ first-strand cDNA synthesis kit (3′ and 5′ adaptor) (Cat# AS-FS-003, Arraystar, Rockville, MD, USA) was used as to synthesize cDNA. Equivalent amounts of cDNA from each donor (in a 1:40 dilution) were mixed with Arraystar SYBR® Green qPCR Master Mix (ROX+) (AS-MR-006-5, Arraystar, Rockville, MD, USA) and loaded onto the 384-well PCR array. As controls, we included: genomic DNA contamination control (GDC), spike-in, positive (PPC) and blank controls. The cycling conditions were: 95°C for 10 min, 40 cycles of 95°C for 10 s and 60°C for 1 min, followed by melting curve analysis. The ΔΔCt method was used to analyze the results. Raw cycle threshold (CT) values for each RNA fragment were normalized to the average of the CTs of the housekeeping genes SNORD43 and SNORD45. CT values over 35 or undetectable were set to 35. ΔΔCt was defined as: ΔCt (PWID) – ΔCt (controls). Fold-difference for each gene from controls to PWID was calculated as 2–ΔΔCt. The P values were calculated based on two-tailed heteroscedastic Student’s t-test of the replicate 2–ΔCt values for each gene in the control and PWID groups.