Male infertility is an important aspect of animal reproduction, which has a high economic impact on the livestock industry. From rapidly increasing list of different sperm fertility biomarkers, the sperm RNA could serve as a promising diagnostic tool to assess male fertility or could have prognostic value for fertilization and embryo development. The aim of this preliminary study was to compare swim-up and somatic cell lysis buffer (SCLB) procedures for extraction of high-quality ram sperm RNA suitable for downstream molecular biology applications.
A modified TRI REAGENT RT procedure with glycogen and lysis step at 65 °C was carried out in order to extract total RNA. Spectrophotometric measurement of quality and quantity of extracted RNA showed A260/280 ratio 1.8 – 1.9, indicating the absence of contaminants and the amount of RNA 24 ± 3.9 µg (unpurified sperm), 0.9 ± 0.11 µg (swim-up) and 1.5 ± 0.2 µg (SCLB). Sperm RNA quality was further validated by RT-qPCR using primers for WBP2NL and MKRN1 genes. The CD18 and CDH1 markers for leucocytes and endothelial cells, respectively, have been used to check a successfull removal of somatic cells from ram sperm by both procedures. Unlike comparable relative amount of WBP2NL and MKRN1 transcripts among unpurified and swim-up or SCLB purified sperm RNA samples, relative amounts of CD18 and CDH1 transcripts were significantly lower in purified sperm RNA samples (p < 0.001), confirming an effective removal of leucocytes and endothelial cells from sperm by both purification techniques. Further investigations could reveal a potential of sperm RNA as a novel biomarker and promising diagnostic tool to assess ram fertility.
PPROM ist eine schwere Komplikation während der Schwangerschaft mit einem hohen Risiko maternaler und fetaler Morbidität sowie fetaler Mortalität. Welche Faktoren beeinflussen das neonatale Outcome?
