Microvolume spectrometer

Prior to RNA isolation, we purified sperm by filtering the semen samples with a 500-mesh sieve to eliminate cell debris. Then, samples were treated with a somatic cell lysis solution (0.3% Triton X-100 and 0.1% SDS in DEPC-treated H₂O) for 30 min on ice to eradicate somatic cells, followed by microscopic analysis of non-sperm cell contamination. We isolated total RNA from the purified ram sperm (n = 4, for each group) samples using the SanPrep column microRNA miniprep kit (Bio Basic Inc, Canada) with slight modification using the manufacturer's protocols. We added 800 μl of a guanidine–thiocyanate lysis buffer enriched in 20 mM DL-dithiothreitol onto the pellet, and then sperm cells were homogenized by passing the samples through a 26-G needle syringe 20–25 times. After other contaminants were thoroughly removed and total RNA was attached to the membrane, an on-column DNase digestion was carried out to remove any traces of DNA contamination. We evaluated the concentration and integrity of the total RNA samples using a NanoDrop (Colibri Microvolume Spectrometer, Titertek-Berthold, Germany) and a 2100-Bioanalyzer with the RNA 2100 Nano Chip (Applied Biosystems, Carlsbad, CA, USA), respectively.

Genomic DNA from Escherichia coli was obtained from the Culture Collection and Research Center (Food Industry Research and Development Institute, HsinChu, Taiwan) and cultivated in Luria–Bertani (LB) broth in an incubator for 16 h at 37 °C. Genomic DNA extraction was performed at 95 °C for 5 min to denature the proteins. The precise concentrations of genomic DNA and mRNA were measured by a microvolume spectrometer (Titertek-Berthold, Pforzheim, Germany).