Oligo dt18

RNA was extracted from cultured cells with TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s recommendations. Reverse transcription reaction was performed by using AccuPower RT Premix and Oligo dT18 primer (Bioneer, Daejeon, Korea) according to the manufacturer’s recommendations. Real-time PCR was performed in a LightCycler nano System (Roche Applied Science, Mannheim, Germany) by using LightCycler DNA Master SYBR GREEN I (Roche Applied Science). PCR mixtures contained 100 ng of cDNA and 0.5 μM each of forward and reverse primers. The samples were denatured at 95 °C for 10 min, followed by 45 cycles of annealing and extension at 95 °C for 20 s, 60 °C for 20 s, and 72 °C for 20 s. Expression values were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Quantitative real-time PCR products were further confirmed by melting curve analysis. The primer sequences were as follows: TNF-α, forward, 5′-AGTGGTGCCAGCCGATGGGTTGT-3′, and reverse, 5′-GCTGAGTTGGTCCCCCTTCTCCAG-3′; IL-8, forward, 5′-TCCAATTCGGGAGACCTCTA-3′, and reverse, 5′-TAGGCATCACTGCCTGTCAA-3′; IL-6, forward, 5′-GGTACATCCTCGACGGCATCT-3′, and reverse, 5′-GTGCCTCTTTGCTGCTTTCAC-3′; GAPDH, forward, 5’-GGAGCCAAAAGGGTCATCAT-3′, and reverse, 5′-GTGATGGCATGGACTGTGGT-3′.

Total mRNA was extracted from HaCaT cells with TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s recommendations. A reverse transcription reaction was performed with AccuPower RT Premix and Oligo dT18 (Bioneer, Daejeon, Korea), according to the manufacturer’s instructions. Real-time PCR was performed in a LightCycler Nano System (Roche Applied Science, Mannheim, Germany) with LightCycler DNA Master SYBR GREEN I (Roche Applied Science, Mannheim, Germany). The PCR mixtures contained 100 ng of cDNA and 0.5 μM each of the forward and reverse primers. The samples were denatured at 95 °C for 10 min, followed by 45 cycles of annealing and extension at 95 °C for 20 s, 60 °C for 20 s, and 72 °C for 20 s. Expression values were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Quantitative real-time PCR products were further confirmed by melting curve analysis. The analyzed target genes were TNF-α, forward, 5’-AGTGGTGCCAGCCGATGGGTTGT-3’, and reverse, 5’-GCTGAGTTGGTCCCCCTTCTCCAG-3’; IL-8, forward, 5’-TCCAATTCGGGAGACCTCTA-3’, and reverse, 5’-TAGGCATCACTGCCTGTCAA-3’; IL-6, forward, 5’-GGTACATCCTCGACGGCATCT-3, and reverse, 5’-GTGCCTCTTTGCTGCTTTCAC-3’; GAPDH, forward, 5’-GGAGCCAAAAGGGTCATCAT-3’, and reverse, 5’-GTGATGGCATGGACTGTGGT-3’.

Total RNA was extracted by using TRizol reagent (Invitrogen, Calsbad, CA, USA). Complementary DNA (cDNA) was synthesized from 1 μg of isolated total RNA, oligo-dT₁₈, and superscript reverse transcriptase (Bioneer, Daejeon, Rep. of Korea) in a final volume of 20ul. For standard PCR, 1 μl of template cDNA was amplified with Taq DNA polymerase. PCR amplification was performed with 25~35 thermocycles for 30 sec at 95 °C, 30 sec at 55 °C, and 60 sec at 72 °C using human (h) oligonucleotide primers specific for hTβ4 (sense: ACA AAC CCG ATA TGG CTG AG; anti-sense: CCT CCA AGG AAG AGA CTG AA), hNPHP3 (sense: AGC GAA ATA CCA AGC AAT GG; anti-sense: TGG AAG GTT CAC TTC CCA AG), hGAPDH (sense: GAA GGT GAA GGT CGG AGT C; anti-sense: GAA GAT GGT GAT GGG ATT TC). Amplified PCR products were separated by 1.0~1.5% agarose gel electrophoresis and detected on Ugenius 3^® gel documentation system (Syngene, Cambridge, United Kingdom).

Total RNA was isolated from liver tissues using TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s methods. Reverse-transcription response was conducted by using AccuPower RT Premix and Oligo dT18 primer (Bioneer, Daejeon, Korea) according to the manufacturer’s protocols. The PCR mixtures contained 100ng of cDNA and 100pM each of forward and reverse primers. The samples were denatured at 95 °C for 10 min, followed by 45 cycles of annealing and extension at 95 °C for 20 s, 60 °C for 20 s, and 72 °C for 20 s. Real-time PCR was accomplished in a LightCycler nano System (Roche Applied Science, Mannheim, Germany) by using LightCycler DNA Master SYBR GREEN I (Roche Applied Science). Expression values were normalized to β-actin. The primer sequences used are listed below in Table 2:

Total mRNA was extracted from the dorsal skin by using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s recommendations. Reverse transcription reaction was performed with AccuPower RT Premix and Oligo dT18 (Bioneer, Daejeon, Korea), according to the manufacturer’s instructions. qRT-PCR was performed using LightCycler® nano instrument (Roche Applied Science, Mannheim, Germany) with TaqMan® Gene Expression Master Mix (Thermo Fisher Scientific). The qRT-PCR mixes contained 100 ng of cDNA and 1 μl each of FAM™ dye-labeled Taqman® Gene Expression Assay and VIC® dye-labeled Taqman® Endogenous Control (predesigned primers and probes; Thermo Fisher Scientific). The expression values of the target gene and the endogenous gene were simultaneously measured by detecting each of the FAM® dye and the VIC® dye. The measured expression values of each target gene were normalized to the expression values of each endogenous gene by using glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The analyzed target genes are IL-4, IL-5, and IL-13.

Total RNA was extracted by using TRizol reagent (Invitrogen, Calsbad, CA, USA). Complementary DNA (cDNA) was synthesized from 1 μg of isolated total RNA, oligo-dT₁₈, and superscript reverse transcriptase (Bioneer, Daejeon, Rep. of Korea) in a final volume of 20 μl. For standard PCR, 1 μl of template cDNA was amplified with Taq DNA polymerase. PCR amplification was performed with 30 ~ 35 thermocycles for 30 s at 95 °C, 30 s at 55 °C, and 60 s at 72 °C using oligonucleotide primers specific for human TB4 (sense: ACA AAC CCG ATA TGG CTG AG; anti-sense: CCT CCA AGG AAG AGA CTG AA), GAPDH (sense: GAA GGT GAA GGT CGG AGT C; anti-sense: GAA GAT GGT GAT GGG ATT TC) and actin (sense: GTC ACC AAC TGG GAC GAC AT; anti-sense: GCA CAG CCT GGA TAG CAA CG). Amplified PCR products were separated by 1.0–2.0% agarose gel electrophoresis and detected on Ugenius 3^® gel documentation system (Syngene, Cambridge, United Kingdom)^{17 (link)}.