Superprep cell lysis kit for qpcr

The Bio-Rad CFX96 real-time PCR system (Bio-Rad Laboratories, Hercules, CA, USA) and THUNDERBIRD SYBR quantitative PCR (qPCR) Mix (TOYOBO Co. Ltd., Osaka, Japan) were used to quantify the levels of target mRNA and internal control mRNA (glyceraldehyde-3-phosphate dehydrogenase, GAPDH). Total RNA was prepared from cells using a SuperPrep Cell Lysis Kit for qPCR (TOYOBO). The conditions for real-time qPCR were 40 cycles at 94°C for 15 s followed by 40 cycles at 60°C for 60 s. Relative IL6 mRNA expressions were normalized to the level of GAPDH mRNA expression. Primers and their sequences for the qPCR are described in Table 2. For the in vitro experiments, all the experiments were performed in triplicate but also performed twice to ensure replication.

The total RNA was isolated from differentiated SH-SY5Y cells using the SuperPrep Cell Lysis Kit for qPCR (Toyobo, Osaka, Japan) according to the manufacturer’s protocol. The total RNA was converted to cDNA using the SuperPrep RT Kit for qPCR (Toyobo). Primers for the human POMC (Forward 5'-AGCCCGCCCAAGGACAAG, reverse 5'-TGCCCTCACTCGCCCTTCT), NPY (forward 5'-TCCAGCCCAGAGACACTGATT, reverse 5’-AGGGTCTTCAAGCCGAGTTCT), and AGRP (forward 5'-CGTCGCTGCGTAAGGCT, reverse 5'-CAGTAGCAGAAGGCATTGAAGAAG) were purchased from Thermo Fisher Scientific. Primers for human ribosomal protein S18 (RPS18) (Primer Set ID: HA067807) were purchased from Takara Bio, Shiga, Japan. qRT-PCR was performed on the Thermal Cycler Dice Real Time System Single (Takara Bio) using the THUNDERBIRD SYBR qPCR Mix (Toyobo). The Ct value was normalized with the housekeeping gene RPS18 and the relative fold change was computed by the ΔΔCt method.