Sybr green 2

To induce genotoxic stress, the cells (15 × 10⁴ per 35 mm dish) were treated with 50 µM etoposide or 1.5 uM doxorubicin for 40 min. The medium was then replaced for a fresh one, and the cells were left in the CO2 incubator to repair the DNA breaks. Following the 8 h of incubation, the cells were trypsinased and washed with PBS. A sample of 10⁴ cells, thoroughly resuspended in 10 µl PBS, was mixed with 0.5% low melting agarose at 37 °C. One day before the experiment, the microscope slides (Menzel-Gläser, GmbH) were immersed into a hot 1% agarose solution and dried overnight at room temperature. The samples were placed onto the agarose pre-coated slides, and dried for 10 min at + 4 °C. The slides were then incubated in the lysis buffer (2.5 M NaCl, 0.1 M EDTA, 10 mM Tris–HCl, pH 10.0, 1% Triton X-100) for 1 h at + 4 °C followed by incubation with the alkaline unwinding buffer (0.2 M NaOH, 1 mM EDTA pH 10.0) for 20 min in the dark at room temperature. Then, slides were rinsed with ice-cold water, subjected to electrophoresis at 23 V for 30 min in a standard 23 cm chamber in an ice-cold buffer (0.2 M NaOH, 1 mM EDTA), and stained with 1:10,000 SYBR Green II (Sigma-Aldrich). Images were analyzed by Open Comet software. More than two hundred cells per sample were evaluated. Experiments were repeated at least three times showing the same trend.

The DNA content of muscle homogenates (diluted 1:8) was measured with the fluorescent dye Hoechst 33258 against a calf thymus DNA standard (Sigma-Aldrich GmbH, Steinheim, Germany) as mentioned in Rehfeldt and Walther (1997 (link)). The RNA content of muscle homogenates (diluted 1:80) was quantified fluorometrically with SYBR Green II against a calf liver RNA standard (Sigma-Aldrich GmbH, Steinheim, Germany) as published by Oksbjerg et al. (2000 ). DNA and RNA assays were performed in 96-well quartz microwell plates by using a Flx-800-I microplate fluorescence reader (Bio-Tek Instruments Inc., Bad Friedrichshall, Germany). Per animal, three and two technical replicates were performed for DNA and RNA, respectively.

Total RNA from P. vulgaris for RT-PCR RNA was extracted from leaves previously treated with liquid nitrogen using TRI Reagent (Sigma-Aldrich, St. Louis, MO, USA) following the manufacturer’s recommended protocol [45 (link)]. The quality of the purified RNA was verified using gel electrophoresis with a 1% denaturing agarose gel stained with SYBR Green II (Sigma-Aldrich, St. Louis, MO, USA), and the concentrations were measured using a NanoDrop 2000c UV–VIS spectrophotometer (Thermo Scientific, Waltham, MA, USA) at 260 nm. To obtain cDNAs, RNAs were reverse transcribed with an oligo dT primer using an enhanced avian myeloblastosis virus reverse transcriptase enzyme (Sigma-Aldrich, St. Louis, MO, USA), following the manufacturer’s recommendation.
The following components were mixed in a sterile microfuge tube: 2 μL 10× reaction buffer; 4 μL MgCl2, 25 mM; 2 μL deoxynucleotide mix 40 mM; 2 μL oligo-p(dT)15 primer (0.8 μg/μL); 1 μL RNase inhibitor 50 U/μL; 1 μg of total RNA; and PCR-grade water to a final volume of 20 μL. All components were supplied by Sigma-Aldrich. The reaction was incubated at +25 °C for 10 min and then at +42 °C for 60 min. Following the +42 °C incubation, the AMV reverse transcriptase was denatured by incubating the reaction at +99 °C for 5 min, and then the reaction was cooled to +4 °C for 5 min.