Qpcr 2x premix sybr

Total RNA was extracted from dorsal skin using TRIzol (Invitrogen, CA, USA). First-strand cDNA synthesis from the total RNA template was performed with PrimeScript^TM RT master mix (Takara, Tokyo, Japan). The resulting cDNA was subjected to real-time PCR using qPCR 2x PreMIX SYBR (Enzynomics, Seoul, Korea) and a CFX-96 thermocycler (Bio-Rad, Hercules, CA, USA). PCR conditions used to amplify all genes were as follows: 10 min at 95 °C and 40 cycles of 95 °C for 15 s and 60 °C for 30 s. Expression data were calculated from the cycle threshold (Ct) value using the ΔCt method of quantification. GAPDH was used for normalization. Oligonucleotides used for real-time PCR are as follows: murine GAPDH, forward, 5′-AGG TCG GTG TGA ACG GAT TTG-3′, reverse, 5′-AGG TTT GAT TCA GGC AGA TGT T-3′; murine TNF-α, forward, 5′-GAT TAT GGC TCA GGG TCC AAC TCT-3′, reverse, 5′-GGA CAT TCG AGG CTC CAG TGA ATT-3′; murine IL-4; forward, 5′-GGT CTC AAC CCC CAG CTA GT-3′, reverse, 5′-GCC GAT GAT CTC TCT CAA GTG AT-3′; murine IL-6, forward, 5′-TAG TCC TTC CTA CCC CAA TTT CC-3′, reverse, 5′-TTG GTC CTT AGC CAC TCC TTC-3′; murine IL-13, forward, 5′-CCT GGC TCT TGC TTG CCT T-3′, reverse, 5′-GGT CTT GTG TGA TGT TGC TCA-3′; murine IFN-γ, forward, 5′-ATG AAC GCT ACA CAC TGC ATC-3′, reverse, 5′-CCA TCC TTT TGC CAG TTC CTC-3′.

Total RNA was extracted from the cells using TRI reagent (Molecular Research Center, Cincinnati, OH, USA). RNA was reverse-transcribed using oligo dT primer and reverse transcriptase (Promega, Madison, WI, USA; A3800) according to the manufacturer’s instructions. Quantitative PCR was performed using qPCR 2X PreMIX SYBR (Enzynomics, Daejeon, Korea; RT500) on a 7500 Real Time PCR System (Applied Biosystems, Foster City, CA, USA). Data were calculated using the 2^−ΔΔCt method. The values were normalized to β-actin levels. Primers were synthesized according to our previous report [15 (link)]. The Cramp primer sequences used were as follows: 5′-CTACCTGAGCAATGTGCCTTC-3′ and 5-CAGGCCTACTACTCTGGCTGA-3′.

At the end of the experiment days, the test animals were sacrificed; the lesion skin area was collected. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Synthesis of the first complementary DNA (cDNA) strand of the entire RNA template was performed using a Prime Script^TM RT Master Mix (Takara, Tokyo, Japan). The cDNA obtained was subjected to real-time PCR using qPCR 2X PreMIX SYBR (Enzynomics, Seoul, South Korea) and a CFX-96 thermocycler (Bio-Rad, Hercules, CA, USA). The PCR conditions used to amplify all genes were as follows: 30 cycles at 95°C for 10 min, 95°C for 10 s, 60°C for 15 s (IFN-γ, 53°C; IL-2, 54°C), and 72°C for 20 s. Expression data were calculated as cycle threshold (Ct) values using ΔCt through a quantification method. GAPDH was used for normalization. The oligonucleotides used are listed in Table 1.