Faststart dna master plus sybr green 1

Human foreskin fibroblasts, Hs68, were seeded at a density of 8 × 10⁴ cells/6 cm dish under adipogenic induction for seven days or 5 × 10⁵ cells/6 cm dish for the study of UVA radiation exposure. For adipogenic induction study, cells were cultured under adipogenesis differentiation medium for seven days and then total RNA was extracted from the Hs68 fibroblasts using REzol^TM C&T reagent (Protech Technologies, Taipei, Taiwan) according to the manufacturer’s instructions. For the photoaging study, total RNA was extracted from Hs68 fibroblasts after exposure to UVA irradiation (4 h) and post-treatment of adiponectin (12 h). RNA (1 μg) was reverse transcribed using TProfessional basic (Biometra, Göttingen, Germany). The resulting cDNA (equivalent to 20 ng) was used in a StepOnePlus™ Real-Time PCR System with a FastStart DNA Master-PLUS SYBR Green I (Applied Biosystems, Foster City, CA, USA). The primers sequences were listed in Table 1. Each sample was corrected using the mean cycle threshold (C_t) value for GADPH. Relative gene expression was analyzed using the ΔC_t method and expressed as fold change (2^−ΔΔCt) T relative to the expression values in non-stimulated cells.

7F2 osteoblasts were seeded in 6 cm dishes for 24 h of incubation. Then, 7F2 cells were incubated with ADM for 7 days to induce adipogenesis or with MM for osteoblast differentiation. For inflammation evaluation, 7F2 osteoblasts were stimulated with IL-1β (10 ng/mL) to induce inflammatory responses. After treatment, total RNA was extracted using the Trizol reagent (Protech Technology, Taipei, Taiwan) following the protocol to the manufacturer’s instructions. RNA (2 μg) was reverse transcribed using TProfessional Basic (Biometra GmbH, Göttingen, Germany). The cDNA (equivalent to 20 ng) was used in an StepOnePlus™ Real-Time PCR System using FastStart DNA Master-PLUS SYBR Green I (Applied Biosystems, Foster City, CA, USA). The designed primers were shown in Table 2 and all primers using nucleotide sequences present in the PrimerBank database. Each sample was corrected using the mean cycle threshold (CT) value for GAPDH. Relative gene expression was analyzed using the ΔCT method and expressed as fold change (2^−ΔCCT) T relative to the expression values in non-stimulated cells.

Cells were seeded at a density of 2 × 10⁵ cells in 6 cm dishes and incubated for 24 h. Inflammatory responses in RAW264.7 cells were stimulated with LPS (0.5 μg/mL), and the cells were treated with 0.05 μg/mL asta-loaded liposomes for 24 h.
After treatment, total RNA was extracted using Trizol reagent (RiboZol, AMRESCO, Solon, OH, USA) following the protocol of the manufacturer’s instructions. For reverse transcription, 1 μg of the total RNA was converted to first-strand cDNA using a reverse transcription kit (Promega, Madison, WI, USA). The resulting cDNA (equivalent to 20 ng) was used in a StepOnePlus Real-Time PCR System using FastStart DNA Master-PLUS SYBR Green I (Applied Biosystems, Foster City, CA, USA). The designed primers are shown in Table 1, and all primers used nucleotide sequences present in the PrimerBank database. Each sample was corrected using the mean cycle threshold (CT) value for GAPDH. Relative gene expression was analyzed using the ΔC_T method and expressed as fold change (2^−ΔCC_T) T relative to the expression values in nonstimulated cells.