Naoac

Flaxseed flour and refined linseed oil were purchased from A.C.E.F. (Fiorenzuola D’Arda, Italy). Linum usitatissimum seeds p.e. 20% lignans (produced by Fontana) was purchase from Farmalabor (Canosa di Puglia, BT). Absolute ethanol (EtOH abs), ethanol 96% (EtOH), Folin–Ciocalteu reagent, TPTZ (≥98%), Trolox (97%), ABTS (≥98%), HCl, FeCl₃, NaOAc, Na₂CO₃, AcOH, and gallic acid were purchased from Sigma–Aldrich (Milano, Italy).
Chitosan FG90 (deacetylation degree 99.97%, MW 100 KDa, viscosity of 1% wt. solution in 1% acetic acid 110 mPa.s) was produced and characterized by Prof. Riccardo Muzzarelli, Department of Biochemistry, Biology and Genetics–Università Politecnica delle Marche-Ancona, Italy. Vaseline, liquid paraffin, cetomacrogol 1000 and cetostearyl alcohol were purchased from Galeno (Carmignano, Italy). MilliQ system Millipore (Rome, Italy) was used to produce ultrapure water.

Tumour and foetal brain tissue were disrupted using an immersion processor and cells were washed in PBS and pelleted before resuspension in TRIzol (Invitrogen, Carlsbad, CA, USA). After 5min of incubation at RT, 0.2 ml of chloroform (AnalaR, Merck, Whitehouse Station, NJ, USA) was added to each 1 ml TRIzol. The phases were mixed and centrifuged (13000 rpm) for 15 min at 4°C. The upper phase was collected and adjusted to 35% ethanol by adding 100% pure ethanol (AnalaR, Merck, Whitehouse Station, NJ, USA). The solution was applied to an RNeasy column (Qiagen, Germantown, MD, USA) and RNA was purified according to the manufacturer's instructions. The RNA was eluted using 50 µl diethyl pyrocarbonate (DEPC)-treated water and precipitated by the addition of 0.1 volume NaOAc (Sigma, St Louis, MO, USA), 2.5 volume of 100% ethanol and 1 µl glycogen (Ambion, Foster City, CA, USA). After 1 hr at −20°C, the samples were centrifuged (13000 rpm) for 20 min at 4°C. The RNA pellet was washed twice with 80% ethanol and air-dried prior to resuspension in 10 µl DEPC-treated water. RNA integrity was measured with an Agilent 2100 Bioanalyser (Agilent, Santa Clara, CA, USA).

Collected spermatozoal RNA was treated with RNase-free recombinant DNase I (Invitrogen, ThermoFisher Scientific, Waltham MA, U.S.A.) according to the protocol provided by the manufacturer. Shortly, 1 μl 10 DNase I reaction buffer and 1 μl DNase I (1 U/μl) were added per 10 μl of DEPC-treated water used for the re-suspension of the RNA pellet, and RNA samples were incubated for 15 min at room temperature. DNase was inactivated by addition of 1 μl of 25 mM EDTA and heating at 65 °C for 10 min, and was then removed through a second precipitation step. Following the addition of 10 μg Glycogen/mL TRIzol®, the tube was filled with RNase−/DNase-free water to 100 μL. Thereafter, 10 μL 3 M NaOAc (Sigma-Aldrich, Buchs, Switzerland) and 100 μL 100% isopropanol (Sigma-Aldrich, Buchs, Switzerland) were added at 1/10 volume. After incubation of samples at room temperature for 45 min, the washing steps with 75% ethanol (Sigma-Aldrich, Buchs, Switzerland) were twice performed as described above. Finally, the RNA pellet was suspended in 10 μL of RNase−/DNase-free water (Gibco, Life Technologies), incubated for 10 min on ice and right after for 10 min at 55 °C before being used for downstream analysis.

Iron chloride hexahydrate (FeCl₃·6H₂O, >97%, Sigma-Aldrich, Korea), NaOAC (>98.5%, Sigma-Aldrich, Republic of Korea), sodium acrylate (NaAc, 97%, Sigma-Aldrich, Republic of Korea), magnesium chloride hexahydrate (MgCl₂·6H₂O, >99%, Sigma-Aldrich, Republic of Korea), and ethylene glycol ((CH₂OH)₂, >99%, Alfa Aesar, Republic of Korea) were used as received without further purification. Deionized water was obtained using a Millipore Direct-Q UV 3.