High pure isolation kit

In order to evaluate the expressions of Bax and Bcl2 genes, U87MG cells (10⁴ cells) were treated with CA (8 μg/mL) individually or in combination with DOX (5 μg/mL). After 24 h treatment, total RNA of cells (7 × 10⁵ cells) was extracted using high pure isolation kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions. Total RNA was assessed qualitatively and quantitatively using a spectrophotometer (NanoDrop™ 2000, USA) and samples stored at -80 °C for further investigations. A one-step quantitative test was performed on RNA expression using CYBR Green kit. The primer sequences were completely similar to those performed in our previous study (20 ). The cDNA synthesis was carried out at thermal cycler conditions corresponding to 15 min at 50 °C, 10 min at 95 °C followed by 40 cycles of 15 s at 95 °C to denature DNA and 45 s at 60 °C to anneal and extend the template (20 ). The melting curve was analyzed at temperatures in range of 65-95 °C with a temperature transient rate of 0.1 °C/s. All reactions were performed in triplicate in a Stratagene MX 3000P system (USA). The values obtained for the target gene expression were normalized to β-actin and analyzed by the relative gene expression 2^-ΔΔCT method (20 ) using the following equation:
-ΔΔCT = (CT target - CT β-actin) Unknown - (CT target - CT β-actin) calibrator
where, CT is threshold cycle.

β-TC₃ cells (2 × 10⁶ cells/well) were seeded in 6 well plates and allowed to adhere for 24 h and then treated with 200 μL of STZ alone or with INF (0.5 mg/ml). Plates were then incubated for 24 h at 37°C in humidified atmosphere of 95% air and 5% CO2. Total DNA from β-TC₃ cells was extracted using high pure isolation kit (Roche, Mannheim, Germany) according to the manufacturer instruction. 20 μl of DNA eluted sample was mixed with 4 μl of novel juice dye and run on 1.2% agarose gel at 75 V for 1 h and visualized using an ultraviolet transilluminator and then photographed.[26 (link)]