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Comments Close. The Comment you have entered exceeds the maximum length. Submit Cancel. Comments Please Post Your Comment. No comments yet. Save Note Note. Double helix is the description of the structure of a DNA molecule. A DNA molecule consists of two strands that wind around each other like a twisted ladder. Each strand has a backbone made of alternating groups of sugar deoxyribose and phosphate groups.
Semen samples were obtained by masturbation from three year-old healthy men. All donors were fertile according to the data given in the sperm donor questionnaire. Complete ejaculate samples were collected in sterile containers. All subjects gave written informed consent in accordance with the Declaration of Helsinki.
The extracellular dsRNA was enriched and purified from the semen using fractionation by equilibrium centrifugation in a CsCl-ethidium bromide density gradient.
A similar approach has been previously used in our laboratory for the purification of dsRNA of a Drosophila reovirus-like virus with a segmented genome Pasyukova and Mukha, To remove the cells and cell debris from the semen, we used two different approaches.
In the first approach, CsCl was added to the freshly collected semen to give a saturated salt solution. Under these conditions, proteins were denatured and an amorphous mass was generated that contains all cellular material. The supernatant was carefully collected for subsequent assays.
The supernatant seminal plasma was carefully collected, and CsCl was added to saturation. The nucleic acids isolated appear identical from the two methods.
Samples saturated with CsCl were diluted to a refractive index 1. After centrifugation, two fractions corresponding to linear DNA generating a band in the middle of the tube and a pellet on the bottom of the tube were observed under UV light nanometers.
A broad diffuse fraction of nucleic acid was also observed between the banding DNA and pellet. A schematic representation of the detected fractions is shown in Figure 1A.
Figure 1. Double stranded RNA from human seminal plasma. A Schematic representation of UV-visible fractions after equilibrium centrifugation of the human seminal plasma from CsCl-ethidium bromide density gradients. Nucleic acids isolated from seminal plasma of two different individuals are shown in lanes with even and odd numbers, respectively.
The fraction located near the top of the gel was digested with DNase I treatment. Each sample was spiked with an in vitro synthesized ssRNA, see arrow, as an internal control, treated with different nucleases, and then separated by electrophoresis and visualized. D Electrophoretic separation of seminal plasma nucleic acids 1 without and 2 after treatment with RiboShredder RNase Blend.
All RNAs in the sample were completely degraded. Electrophoretic separation of the semen plasma nucleic acids after DNase I treatment is shown in Figure 1B , lanes 3 and 4 two different donors , and after treatment by both DNase I and RNase One Ribonuclease in Figure 1B , lanes 5 and 6 two different donors.
The fraction located near the top of the gel Figure 1B , lanes 1 and 2 was digested with DNase I treatment, indicating that this fraction is represented by DNA molecules.
The origin of the dsRNAs in seminal fluid remains unclear. In summary, we have shown that human seminal plasma contains a repertoire of cell-free dsRNA. We do realize that the identity, source, and functioning of these dsRNA are yet undetermined. We hypothesize that these dsRNAs could influence the implementation of genetic information or gene regulation in offspring and, if future sequencing reveals polymorphisms in dsRNA nucleotide composition among individuals, characterization of seminal fluid dsRNAs might be potentially useful as non-invasive molecular markers.
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