RNA purification

Total RNA (contains small RNAs, including miRNAs) was isolated from sperm using an RNeasy plus Universal Mini Kit (Qiagen Inc., Valencia, CA, USA), in accordance with manufacturer’s instructions. Briefly, 900 μL QIAzol Lysis Reagent was added to the sperm pellet (~500 × 10⁶ sperm) and thoroughly homogenized using a disposable homogenizer (Thermo Fisher Scientific, San Francisco, CA, USA). The homogenate was placed at room temperature for 5 min to promote dissociation of nucleoprotein complexes. Then, 100 μL of genomic DNA (gDNA) eliminator solution was added and the mixture shaken vigorously to eliminate contamination by gDNA. Chloroform (180 μL) was then added to the homogenate. After vigorous shaking and 2–3 min incubation at room temperature, the mixture was centrifuged at 12,000 × g at 4 °C for 15 min. After centrifugation, the upper aqueous phase (~600 μL) was transferred to a new microcentrifuge tube and 1.5 times volume of 100% ethanol was added. The mixture was mixed thoroughly by repeated pipetting and the sample was centrifuged in an RNeasy mini spin column (8,000 × g for 15 s at room temperature). The RNA was bound to the membrane of the spin column and subsequently removed using buffer RWT and buffer RPE by centrifugation. Thereafter, RNA was eluted in 60 μL RNase-free water. Purity of the RNA was determined using a Thermo Scientific NanoDrop 1000 spectrophotometer; the ratio of absorbance at 260 and 280 nm, respectively, was ~2.0 for all samples. All RNA samples were stored at −80 °C until used.