Kapa sybr green pcr master mix

cDNA was synthesized from the isolated total RNA (Nippon Genetics, Duren, Germany), and the quantitative real-time PCR (qPCR) method (Kapa SYBR^® Green PCR Master Mix, Sigma-Aldrich, St Louis, MO, USA) was used to measure HIF1A, HMOX1, EPO, ISG15, LDHA, HK2, and VEGFA mRNA expression using the specific primer sets listed in Table 1.
Quantitative real-time PCR was performed in 96-well plates (CFX Connect thermocycler, Biorad, Hercules, CA, USA). The comparative CT method 2^−ΔΔCT [33 (link)] was applied using GAPDH mRNA expression levels for normalization of the target gene expression levels. ICU admission values within each group were used as the control.

Total RNA was extracted using the RNeasy Mini Kit (Qiagen, 74106, Hilden, Germany) according to the manufacturer’s instructions. cDNA was prepared from RNA extracts using the PrimeScript RT Reagent Kit (Takara, RR037A, Kusatsu shi, Japan) according to the manufacturer’s instructions. The expression of selected genes was analyzed by quantitative real-time PCR performed on a CFX96 C1000 Thermocycler (BioRad) using Kapa SYBRgreen PCR master mix (Sigma, KK4617) as previously described [47 (link)]. The expression of the different analyzed genes was normalized to the expression of β-actin. The primer sequences are given in Table 1.