Sybr gold

DNA was extracted from the tissue section using the NucleoSpin Tissue for DNA, RNA and protein purification kit (Macherey–Nagel, USA) according to the manufacturer’s protocol. The amplification of each of the 10 exons was performed by PCR using the specific primers listed in Additional file 1: Table S1. The oligonucleotide primers were designed to span the entire exon and a sufficient flanking intron sequence to include the splice junction mutations for the analysis. SSCP was performed as follows: 100 ng of DNA was amplified for 35 cycles using the 5× FIREPol PCR mastermix ready to load (SOLIS BIODYNE, Estonia). The PCR product was denatured by heat shock and was run on a 12% polyacrylamide gel at 160 V for 2 h. The gel was stained by SYBR gold (Qiagen Inc.) for 15 min and visualized using the UVP BioImaging system. The wild type p53 gene in the MCF-7 cell line was used as a control. All the samples that showed altered mobility were further characterized by DNA sequencing for the altered exon.

First, 400,000 cells per well were seeded on a 12-well plate overnight, treated accordingly, harvested, and resuspended in 100 µL PBS. Then, 20 µL of this suspension was mixed with low-melting agarose (Trevigen, Gaithersburg, MD, USA), spread evenly over CometSlides (Trevigen, Gaithersburg, MD, USA), left to congeal, and then kept in lysis solution (Trevigen) at 4 °C overnight. Thereafter, the slides were immersed in unwinding solution for 30 min at room temperature before gel electrophoresis was run for 25 min. The slides were washed, dried at 37 °C overnight, and stained with SYBRGold (Qiagen, Hilden, Germany). Fluorescence images were taken with Olympus Fluoview FV1000 confocal microscope (Olympus, Tokyo, Japan) and analysed using OpenComet [93 (link)]. The tail moment and the olive moment were calculated as follows: