4.4. Analysis of the Expression of ER Stress Markers

Total RNA was isolated from HTM cells using the PureLink RNA Mini Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) according to the manufacturer’s protocol. Isolated RNA was transcribed into cDNA to a final concentration of 100 ng using GoScript ™ Reverse Transcriptase (Promega Inc., Madison, WI, USA) according to the manufacturers protocol. Subsequently, we performed the TaqMan Gene Expression Assays to analyze the expression profile of genes related to ER stress conditions. For this purpose, we evaluated the level of the expression of three genes: ATF4 (Hs00909569_g1), DDIT3 (Hs01090850_m1) and BAX (Hs00180269_m1). As a reference gene, the ACTB (Hs99999903_m1) was used. For the qPCR analysis, the total reaction volume was 10 µL, that includes the following reagents: 1 uL cDNA, 1 µL primers, 2 µL 5x HOT FIREPol^® Probe qPCR Mix (Solis BioDyne, Tartu, Estonia) and 6 µL nuclease free water. Reaction conditions were used regarding to the manufacturers protocol: enzyme activation (15 min, 95 °C), denaturation (40 cycles, 10 s, 95 °C), annealing/extension (40 cycles, 60 s, 60 °C). Gene expression was performed using the Bio-Rad CFX96 (BioRad, Hercules, CA, USA) system.