Total rna mini kit

Total RNA was extracted from the ASCs cells at the end of the differentiation process using Total RNA mini kit (Favorgen, Taiwan). The purity and concentration of total RNA were assessed by NanoDrop spectrophotometer (Thermofisher Scientific, USA). RNA samples were reverse‐transcribed using cDNA synthesis Kit (PCR Biosystems, UK) according to the manufacturer's instruction. Specific primers, shown in Table 1, for amplification of Keratin‐10 (KRT10), Involucrin, collagen type I (Collagen‐I), Vimentin, and GAPDH were used to quantify the expression of differentiation markers by real‐time PCR.³¹ The quantitative PCR reaction was performed using SYBR Green PCR Master Mix (PCR Biosystems, UK) in a Step One instrument (Applied Biosystems, USA). The PCR reaction was carried out by adding 2 μl of cDNA in 10 μl SYBR Green PCR Master Mix and 200 nM of each primer per reaction. RNase/DNAse‐free water was added to obtain 20 μl final volume. The amplification process was performed in one cycle of 95°C for 2 min, followed by 40 cycles of 95°C for 5 s, 60°C for 25 s, and the final thermal denaturation steps for melting curve analysis. ASCs without treatment were used as negative controls. Relative target gene expression levels in entire samples were calculated based on the 2^−ΔΔCT method considering GAPDH as the internal control gene.

3T3-L1 cells were seeded at 2 × 10⁴ cells/well in a 24-well plate and cultured with the conditioned media containing CQ-H at a concentration of 80 µg/mL for 8 days. Total ribonucleic acid (RNA) was extracted using the total RNA mini kit (Favorgen, Ping Tung, Taiwan; cat#: FATRK001). Complementary DNA was synthesized using the iScript reverse transcription supermix (Biorad, Hercules, CA, USA; cat#: 1708841) following the manufacturer’s protocols. Quantitative polymerase chain reaction (PCR) was performed with the iTaq Universal SYBR Green Supermix (Biorad, Hercules, CA, USA; cat#: 1725122) using the Applied BiosystemsTM 7500 real-time PCR system. Melting curve analysis was performed to confirm the amplification of the target gene. The gene names and corresponding primer sequences are listed in Table 2. Gene expression levels were determined using the 2^−ΔΔCT method with 18s normalization, and a cut-off of two-fold change was applied [30 (link)].

Total RNA was isolated using a Total RNA mini kit (Favorgen Biotech, Ping-Tung, Taiwan) according to the manufacturer’s instructions. Two micrograms of total RNA were used for cDNA synthesis and qPCR was performed on a Rotor-Gene Q 5plex (QIAGEN; located at the Korea Basic Science Institute in Gwangju, Korea) using the QuantiTect SYBR Green PCR Kit (QIAGEN, Hildon, Germany). The following primers were used for qPCR: Nfatc1 5′- CTCGAAAGACAGCACTGGAGCAT-3′ (forward) and 5′-CGGCTGCCTTCCGTCTCATAG-3′ (reverse); Oscar 5′-CTGCTGGTAACGGATCAGCTCCCCAGA-3′ (forward) and 5′-CCAAGGAGCCAGAACCTTCGAAACT-3′ (reverse); Ctsk 5′-ACGGAGGCATTGACTCTGAAGATG-3′ and 5′-GGAACCACCAACGAGAGGAGAAAT-3′ (reverse); Trap 5′-CTGGAGTGCACGATGCCAGCGACA-3′ (forward) and 5′-TCCGTGCTCGGCGATGCACCAGA-3′ (reverse); c-Fos 5′-CCAGTCAAGAGCATCAGCAA-3′ (forward) and 5′-AAGTAGTCGCAGCCCCGAGTA-3′ (reverse); Ctr 5′-TGGTTGAGGTTGTGCCCA-3′ (forward) and 5′-CTCGTGGGTTTGCCTCATC-3′ (reverse); Mmp-9 5′-CGTCGTGATCCCCACTTACT-3′ (forward) and 5′-AACACACAGGGTTTGCCTTC-3′ (reverse). The mean Ct value of three determinations for each gene was divided by the linear Ct of the β-actin (Actb) gene to obtain the relative abundance of the transcript. Mean values were obtained from three or four separate experiments. Actb was used as an internal control for all measurements.