Takara sybrr premix ex taqtm 2

Total RNAs were extracted using Trizol reagent (Invitrogen, Carlsbad, United States), then were dissolved in nuclease-free water, followed by measurement of RNA concentrations using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Shanghai, China). Complementary DNA was reverse transcribed from each total RNA using a Takara PrimeScript^TM RT reagent kit (Takara Bio Inc, Shiga, Japan). PCR reactions were performed with Takara SYBR^R Premix Ex Taq^TM II (Takara Bio Inc.) using a LightCycler^R 480 II (Roche Diagnostics, Rotkreuz, Switzerland). One hundred ng of each cDNA in a total of 20 µl qRT-PCR reaction was used for amplification. PCR reactions were carried out at 95 °C for 30 s followed by 40 cycles at 95 °C for 5 s and at 60 °C for 20 s and ended with an elongation step for 15 s at 72 °C. Ct values were used for quantification and relative mRNA expression levels were calculated by the 2^-ΔΔCt method normalized by the human housekeeping gene 36β4. PCR primers used are listed in Table S1.

Total RNAs were extracted from cells using an RNA Extraction kit (Takara Bio Inc., Kusatsu, Shiga Prefecture, Japan) according to the protocol of the manufacturer. The RNAs were dissolved in nuclease-free water and the concentrations were measured using a NanoDrop spectrophotometer. One μg of each total RNA was reverse transcribed to complementary DNA (cDNA) using a Takara PrimeScriptTM RT kit (Takara Bio Inc.). PCR reactions were performed with Takara SYBRR Premix Ex TaqTM II (Takara Bio Inc.) using a LightCyclerR 480 II (Roche Diagnostics Ltd., Rotkreuz, Switzerland). One hundred ng cDNA and 250 nmol gene specific primers were used for amplification in 20 μl qRT-PCR reactions. The PCR reactions were carried out at 95°C for 30 s followed by 40 repeated cycles of 95°C for 5 s and 60°C for 20 s and then were terminated with an elongation step for 15 s at 72°C. Data were acquired and analyzed with a LightCycler 480 system. The PCR primers used are listed in S1 Table.

Total RNA was isolated from primary human keratinocytes and skin tissues using an RNAeasy kit (Cat. 74104, Qiagen, Germantown, MD, USA) after which the total RNA was reverse transcribed into cDNA using a superscript III first strand kit (Cat. 1708891, Bio-Rad, Hercules, CA, USA). Takara SYBRR Premix Ex TaqTM II (Cat. RR820B, Takara Bio Inc., Mountain View, CA, USA) was used in the PCR procedure with the LightCyclerR 480 II (Roche Diagnostics, Risch-Rotkreuz, Switzerland). Following that, qRT-PCR amplification was performed in a 20 µL volume with 100 ng cDNA. The PCR procedure was as follows: 30 s at 95 °C; 40 cycles of 5 s at 95 °C, 20 s at 60 °C and 15 s elongation at 72 °C. Furthermore, Ct-values were calculated to quantify mRNA expression levels by 2^−ΔΔCt, with 36β4 serving as the endogenous reference. The oligo sequences for genes used in PCR are shown in Supplementary Table S1.