Thermo scientific nanodrop 2000c spectrophotometer

Twenty-five milligrams of umbilical cord pooled from each litter were weighed and incubated in enzymatic tissue digestion solution [50 mM Tris-HCl, 100 mM EDTA, 100 mM NaCl, 1% SDS pH 8.0, and 0.5 mg/ml of proteinase K (Invitrogen)] at 50°C for 3 h, and then homogenized. DNA and RNA extraction of tissue samples was performed using 500 µl/sample of TRIzol reagent (Invitrogen) following manufacturers’ instructions. The nucleic acid quality was assessed by measuring the absorbance at 260/280 nm and was quantified with a Thermo Scientific Nanodrop 2000c Spectrophotometer (Thermo Scientific). The integrity of DNA and RNA was verified by 1% agarose gel electrophoresis and ethidium bromide staining. Isolated DNA and RNA samples were preserved at −76°C until examination.

Optical rotations were measured on a Rudolph research analytical AUTOPOL IV automatic polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA), IR spectra were recorded with a Bruker ALPHA II FT-IR spectrometer (Bruker, Billerica, MA, USA), and UV spectra were measured with a Thermo Scientific Nanodrop 2000C spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). High-performance liquid chromatography (HPLC) was performed using a Varian ProStar 215 solvent delivery module equipped with a Varian Prostar 320 UV-Vis detector (Agilent Technologies, Santa Clara, CA, USA), operating under Star 6.41 chromatography workstation software (6.41, Agilent Technologies, Santa Clara, CA, USA). NMR spectra were obtained with a Bruker Avance III NMR spectrometer (Bruker, Billerica, MA, USA) equipped with a 3 mm cryogenic probe and operating at 600 MHz for ¹H and 150 MHz for ¹³C. Spectra were calibrated to residual solvent signals at δ_H 2.50 and δ_C 39.5 (DMSO-d₆), and δ_H 3.31 and δ_C 49.0 (CD₃OD). All 2D NMR experiments were acquired with nonuniform sampling (NUS) set to 50% or 25%. HMBC experiments were run with ⁿJ_CH = 8.0, 3.5, or 2.0 Hz, and the LR-HSQMBC experiment was optimized for ⁿJ_CH = 2.0 Hz. HRESIMS data were acquired on an Agilent Technology 6530 Accurate-mass Q-TOF LC/MS (Agilent Technologies, Santa Clara, CA, USA).

Total DNA from 50 mg of umbilical vein was extracted as follows: enzymatic tissue digestion was carried out with 500 μl/sample of solution [50 mM Tris-HCl, 100 mM EDTA, 100 mM NaCl, 1% SDS pH 8.0 and, 0.5 mg/ml of proteinase K (Invitrogen)] and it was incubated at 50°C overnight. The following day, the digested sample was centrifugated at 2,400 x g during 5 min to rescue the pellet in a new DNasa/RNasa-free microfuge tube and homogenized in 500 μl of TRIzol reagent (Invitrogen), and incubated at room temperature for 5 min. Ten μl of chloroform was added, mixed, incubated at room temperature for 3 min and, centrifuged at 11,200 x rpm for 15 min at 4°C. DNA precipitation was followed by addition of 100% ethanol and centrifugation at 4,700 x g for 5 min at 4°C. DNA pellet was rinsed three times, adding 500 μl of sodium citrate 0.1M/10% ethanol, incubated at room temperature 30 min and, centrifuged at 2,000 x g for 5 min at 4°C. Last rinsing was made with 400 μl of 75% ethanol, incubated 15 min and centrifuged. DNA was dissolved in 25 μl of TE buffer and incubated at 45°C for 1 h. DNA quality was assessed by measuring the absorbance at 260/280 nm (1.7–2.0) and was quantified with a Thermo Scientific Nanodrop 2000c Spectrophotometer (Thermo Scientific). DNA integrity was verified by 1% agarose gel electrophoresis.