2.4. RNA extraction, cDNA synthesis and qRT‐PCR

RNA was extracted by peqGOLD Total RNA Kit (PEQLAB Biotechnologie GmbH, VWR) and on‐column DNase digestion (RNase‐free DNase set; Qiagen VWR). To determine RNA concentration and to assess the purity, a NanoDrop 1000 spectrophotometer (Thermo Scientific) was used. cDNA was prepared using at least 1 μg of mRNA and maxima first strand cDNA synthesis Kit, according to manufacturer instructions (Thermo Fischer Scientific). qPCR reactions were performed by adding 5 μl of cDNA, 7.5 μl of SYBR Green PCR Master Mix (Applied Biosystems), and 0.2 μM of primers mix. The reactions were performed using a QuantStudio 5 Real‐Time PCR System (Thermo Fisher Scientific) and results were analyzed by Applied Biosystems QuantStudio™ 3 & 5 Real‐Time PCR System software. Amplification conditions were proceeded as hold stage at 2 min at 50°C, 10 min at 95°C, 40 cycles of 15 seconds at 95°C and 1 min at the annealing temperature. The specificity of the qRT‐PCR reactions was confirmed by melting curve analyses performed at the final dissociation stage. The primers used are described in Table 1.

Primer set used for qPCR

The expression values were normalized against the house‐keeping gene hypoxanthine‐guanine phosphoribosyltransferase (HPRT) and the relative change in gene expression was analyzed and calculated according to the 2^ΔΔCt method.