Quantstudio 3 real time pcr system

The changes in the expression of IL-4R⍺, H4R, TRPV1, TRPV4, and TRPM8 in NHEK and organotypic 3D skin were examined. YWHAZ was used as a reference gene to normalize the mismatch in mRNA quantity. The sequences of the individual primers used in the experiment are listed in Table S1. The qPCR was performed with the QuantStudio™ 3 Real-Time PCR System using the FastStart Essential DNA Green Master Kit (Roche Diagnostics, Basel, Switzerland, cat. no. 06402001). For each reaction, 5 ng of cDNA was given and qPCR was performed in triplicate as follows: Denaturation at 95°C for 10 min, amplification and quantification were repeated 45 times (95°C for 20s, 60°C for 20s, and 72°C for 20s with a single fluorescence measurement), melting curve at 60–95°C with a heating rate of 0.1°C per second and continuous fluorescence measurement, final cooling to 4°C. A negative control without cDNA and an inter-run calibrator were included in each assay. Gene expression was analyzed according to Pfaffl.46 (link) Results were scaled by the expression level of the control, which was set as one.

The changes in the expression of TLR2, NF-κB and COX-2 were investigated. Tyrosine 3-Monooxygenase (YWHAZ) was used as a reference gene to normalize the disproportion in mRNA quantity. The oligonucleotide primers specific for each gene are listed in Table S1. The qPCR was performed in the QuantStudio™ 3 Real-Time PCR System using the FastStart Essential DNA Green Master Kit (Roche Diagnostics, Basel, Switzerland, cat. no. 06402001). For each reaction, 5 ng of cDNA was given and qPCR was performed in triplicates as follows: denaturation at 95°C for 10 min, amplification and quantification repeated 45 times (95°C for 20s, 60°C for 20s and 72°C for 20s with a single fluorescence measurement), melting curve at 60–95°C with 0.1°C per second heating rate and continuous fluorescence measurement, final cooling to 4°C. A negative control without cDNA and an inter-run calibrator were included in each assay. Gene expression was analyzed according to Pfaffl.37 (link) Results were scaled to the expression level of the control, which was determined as one.