Maxima sybr green qpcr master mix kit

A qPCR assay was carried out to examine the Hsp gene mRNA levels; the primers used are shown in Table 1. qPCR was performed using an ABI 7300 real-time PCR detection system (ABI, USA) and Maxima SYBR Green qPCR Master Mix kit (TaKaRa, Dalian, China). The relative Hsp gene mRNA levels were assessed using the following cycling parameters: an initial denaturation at 95°C for 3 min followed by 40 cycles of 95°C for 20 s, 55°C for 20 s and 72°C for 20 s. After PCR, the homogeneity of the PCR product was confirmed by melting curve analysis. β-actin was used as an internal control, and the ratios of Hsps/β-actin mRNA expression were calculated. The fold changes in all experiments were calculated according to the 2^-ΔΔCt method [18 (link)]. qPCR was repeated three times for each gene. Each replication comprised two technical replicates.

Total RNA was extracted from the cultured cells using TRIzol reagent (Invitrogen, Carlsbad, CA, United States), according to the manufacturer’s instructions. RNA was quantified using a NanoDrop 2000 spectrometer (Thermo Fisher Scientific, United States). Reverse transcription was performed using 2 mg RNA at 42°C for 50 min with a mixture containing 200 U SuperScript II reverse transcriptase enzyme, 125 ng random primers, and 0.5 mM dNTP Mix (Invitrogen, United States).
Real-time PCR validation was conducted using the Maxima® SYBR® Green qPCR Master Mix kit (Takara Bio, Dalian, China), according to the manufacturer’s instructions, in an ABI Prism 7500 Sequence Detection System 288 (Applied Biosystems Inc.). The following cycling condition were employed: 95°C for 10 min; 95°C for 30 s, 60°C for 1 min, and 72°C for 1 min; 40 cycles. GAPDH was used as the standardized internal control. The primer sequences are listed in Table 1. Each sample was repeated in triplicate and the fold change in gene expression was calculated according to the 2^-ΔΔCt method.