Thermo maxima sybr green qpcr master mix

The mRNA expression levels of DEFB4, KRT1, KRT17, GLUT1 and ACTB were measured by real-time qRT-PCR. Total RNA was extracted from HPK using the TRIzol reagent (Thermo Fisher, Darmstadt, Germany). After extraction, 1 µg of total RNA was reverse transcribed using the iScript cDNA Synthesis Kit (BIO-RAD, Feldkirchen, Germany) to obtain cDNA. The real-time qRT-PCR reaction was performed using a SYBR Green kit (Thermo Maxima SYBR Green qPCR Master Mix, Thermo Fisher, Darmstadt, Germany) on a Bio-RAD CFX96^TM Real-Time System. The used primers were the following:
Hu DEFB4 5′-ACCACCAAAAACACCTGGAAG-3′ forward and 5′-ACCAGGGACCAGGACCTTTA-3′ reverse; hu KRT1 5′-GGCAGACATGGGGATAGTGTG-3′ forward and 5′-CTTGAGGGCATTCTCGCCA-3′ reverse; hu KRT17 5′-GAGATTGCCACCTACCGCC-3′ forward and 5′-ACCTCTTCCACAATGGTACGC-3′ reverse; hu GLUT1 5′-TCTGGCATCAACGCTGTCTT-3′ forward and 5′-AAGGCAAGTGTCTCGACAGG-3′ reverse; hu ACTB: 5′-CACTGTCGAGT CGCGTCC-3′ forward and 5′-TCATCCATGGCGAACTGGTG-3′ reverse. The relative gene expression was determined using the comparative C_T method using the formula of 2^−ΔΔCt [50 (link)] with ACTB as the internal control. Data were expressed as mean ± standard deviation (SD) of three independent experiments from 3 different skin biopsies.

The total RNA was extracted from frozen villi samples using Trizol reagent, and its purity was evaluated using a Nanophotometer (Implen, Germany). Following reverse transcription with the Thermo RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, United States), 10μl cDNA was amplified using Thermo Maxima SYBR Green qPCR Master Mix (Thermo Fisher Scientific, United States) on the CFX96 cycler (Bio-rad, United States). The RT-PCR parameters were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 10 s, 60°C for 34 s, and 95°C for 15 s. All primers were designed and synthesized by Sangon Bioengineering Company (Table 1). The expression levels of the target genes were calculated by the (2^–ΔΔCT) method.