Presence and Characterization of Cell-Free Seminal RNA in Healthy Individuals: Implications for Noninvasive Disease Diagnosis and Gene Expression Studies of the Male Reproductive System

Abstract

Abstract Background: We recently detected cell-free seminal RNA (cfsRNA) and set out to study its concentration, integrity, stability in healthy individuals, and mechanisms for its protection from ribonucleases. Methods: We quantified cfsRNA by reverse-transcription quantitative real-time PCR (RT-qPCR) targeting of the 5′ region of the ACTB (actin, beta) transcript. cfsRNA integrity was analyzed by microcapillary electrophoresis and by amplification of full-length ACTB and DDX4 [DEAD (Asp-Glu-Ala-Asp) box polypeptide 4] transcripts, including measurement of the relative amounts of different regions of ACTB and DDX4 transcripts. Stability of cfsRNA was measured by time-course analysis of different regions of ACTB and DDX4 transcripts. To investigate whether cfsRNA was protected in complexed forms, we processed seminal plasma in 2 ways: filtration through pores of different sizes and Triton X-100 treatment before RNA recovery. Results: cfsRNA concentrations varied from 0.87–3.64 mg/L [mean (SD), 1.75 mg/L (0.92 mg/L)]. Most cfsRNA was present in partially degraded forms, with smaller amounts of middle and 3′ amplicons compared with 5′ amplicons. Although the 3′ region of the DDX4 transcript was degraded completely by 90 min, the 5′ regions of ACTB and DDX4 transcripts were stable up to 24 h. Filtration through 0.22-μm pores reduced ACTB and DDX4 mRNA concentrations by 72% and 61%, respectively. Nearly all seminal ACTB and DDX4 mRNA disappeared after Triton X-100 treatment. Conclusions: Although cfsRNA was partially degraded, it represented diverse transcript species and was abundant, fairly stable, and associated with particles in healthy individuals. cfsRNA may represent a potential noninvasive biomarker of the male reproductive system and of germline epigenetics.