Sg qpcr master mix 2

The following chemicals were obtained from Sigma-Aldrich (Steinheim, Germany): 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Dulbecco’s Modified Eagle Medium (DMEM) with and without phenol red, propidium iodide (PI), Triton X-100, dichloromethane (DCM), hexane (Hx), L-α-phosphatidylcholine (L-α-PC) from chicken’s egg, chloroform, tert-Butyl hydroperoxide solution, cadmium acetate, and deuterium oxide. 5,5-Dimethyl-1-Pyrroline N-oxide (DMPO) was obtained from Dojindo (Kumamoto, Japan). Fetal bovine serum (FBS) was purchased from Gibco (Carlsbad, CA, USA). Potassium iodide was purchased from Chempur (Piekary Slaskie, Poland). Acetic acid and dimethyl sulfoxide (DMSO) were purchased from POCH (Gliwice, Poland). Alexa Fluor 488 Annexin V/Dead Cell Apoptosis Kit was purchased from Life Technologies (Carlsbad, CA, USA). Caspase-Glo^® 3/7 was purchased from Promega (Madison, WI, USA). JC-10 Mitochondrial Membrane Potential Assay Kit was purchased from Abcam (Cambridge, UK). RNA Extracol, NG dART RT kit, and SG qPCR Master Mix (2×) were obtained from EURx (Gdansk, Poland).

The expression level of genes connected with DNA repair and oxidative stress response was evaluated with quantitative real-time PCR (qRT-PCR) method. The A375 cells were seeded in 6-well culture plates at a density 1 × 10⁵ cells/mL and after 70–80% confluency achievement the cells were treated with CuT1, Cu10, CuT16 in concentrations corresponding to IC₅₀ values or DMSO as vehicle in control cultures. After 24h of incubation total RNA was isolated using the Syngen Blood/Cell RNA Mini Kit (Syngen Biotech, Wroclaw, Poland). All samples were transcribed using NG dART RT-PCR reagents (EURx, Gdansk, Poland) according to the manufacturer’s instructions. The relative mRNA expression level was determined by relative quantification method (ΔΔCt) using 18S ribosomal N5 (RNA18SN5) and beta-actin (ACTB) as endogenous controls. The reference genes were selected on the basis of our preliminary studies, where RNA18SN5 and ACTB remained unaffected by the experimental conditions. The PCR was conducted in triplicate using Applied Biosystems^® 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) and SG qPCR Master Mix (2×) (EURx, Gdansk, Poland) in accordance with the manufacturer’s protocol. Data was presented and analysed as RQ values (RQ = 2^−ΔΔCt). The genes and sequences of used primers were listed in Table 5.

Total RNA was isolated from the cultivated Colon26 and HT29 cells, treated with GO nanoparticles in combination with NIR irradiation for 24 h and 72 h, using Universal RNA Purification Kit (EURx), including the optional DNase I digestion step. This was followed by reverse transcription into cDNA of 280 ng DNase I-treated total RNA, using NG dART RT-PCR kit (EURx). Gene expression analysis was performed for the reference gene (GAPDH) and the genes of interest—ATM, TP53, BBC3 (PUMA), CDKN1A (p21), and RAD51. The used primers are described in Table 1. The reaction was carried out by the use of SG qPCR Master Mix (2×) (EURx), with 14 ng total RNA and 0.5 µM primer concentration, on Rotor-Gene 6000 (Corbett LifeScience). Three repetitions of the experiment were performed. The results were analyzed using the comparative CT method (ΔΔCT method) [47 (link)]. More than a 2-fold change in the expression level (up or down) compared to the calibrator (the respective untreated control group) was considered as significant.