Revertra ace qpcr rt master mix

Bergapten and methoxsalen (purity > 98%) were purchased from TCI (Tokyo, Japan). STZ was acquired from Sigma-Aldrich (St. Louis, MO, USA). Many kits were purchased; serum bone-ALP and OCN enzyme-linked immunosorbent assay (ELISA) kits from Elabscience (Wuhan, China), a TRAP ELISA kit from Cusabio Biotech (Wuhan, China), an insulin kit from Morinaga Institute of Biological Science, Inc. (Yokohama, Japan) and an adiponectin kit from R&D Systems, Inc. (Minneapolis, MN, USA). TRIzol reagent, ReverTra Ace qPCR RT master mix and SYBR green PCR kit were acquired from Invitrogen (Carlsbad, CA, USA), Toyobo (Osaka, Japan) and Qiagen (Hilden, Germany), respectively.

Total RNA was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and 1 μg of total RNA was reverse-transcribed using a ReverTra Ace^® qPCR RT Master Mix (Cat. No. FSQ-201; Toyobo Co., Okaka, Japan) according to the manufacturer’s instructions. Real-time PCR was performed using iQ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. The threshold cycle (C_T) was determined using Bio-Rad CFX manager 3.1 software. Relative expression levels between compounds and untreated controls normalized to the levels of β-Actin mRNA were calculated using the comparative C_T method. All experiments were performed in quadruplicate, and the sequences of primers (Bioneer, Daejeon, Korea) are as follows: β-actin forward, 5′-AGC ACA ATG AAG ATC AAG AT-3′; β-actin reverse, 5′-TGT AAC GCA ACT AAG TCA TA-3′; COX-2 forward, 5′-CTT CAC GCA TCA GTT TTT CAA G-3′; and COX-2 reverse 5′-TCA CCG TAA ATA TGA TTT AAG TCA AC-3′.