Sybr premix ex taq 2 system

Total RNA of the cells was isolated using RNAfast 200 reagent (Shanghai Fastagen Biotechnology Co., Ltd., Shanghai, China) and quantitated by measuring the absorbance at a wavelength of 260 nm. The RNA (2 µg) sample was reverse transcribed with 5X PrimeScript RT Master Mix (2 µl; Takara Biotechnology Co., Ltd., Dalian, China), RNase-free dH₂O (7 µl) and total RNA (1 µl) were mixed and reacted at 37°C for 16 min and 85°C for 5 sec. qPCR was then performed using the SYBR Premix Ex Taq™ II system (Takara Biotechnology Co., Ltd.) and the Bio-Rad CFX96™ Real-time system (Bio-Rad Laboratories, Inc.). SYBR Premix Ex Taq II (12.5 µl), 1 µl sense primer (10 µM), 1 µl anti-sense primer (10 µM) 2 µl cDNA solution and 8.5 µl RNase-free water were mixed together. The following thermocycling protocol was used with three stages, including pre-degeneration for 95°C for 30 sec, one repeat; PCR amplification at 95°C for 5 sec followed by 60°C for 30 sec, 40 repeats; and dissociation at 95°C for 15 sec followed by 60°C for 30 sec and 95°C for 15 sec. GAPDH was used as the loading control to balance the quantity of the samples, and the gene expression was normalized to the GAPDH to calculate relative expression level using the 2^−ΔΔCq method (18 (link)). The gene-specific primers used are listed in Table II.

For RT-qPCR, total RNA was isolated from INS-1 cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA was synthesized using a PrimeScript^® RT Master Mix kit (Takara Bio, Inc.), according to the manufacturer's protocols. The resulting cDNA was used for qPCR analysis using an SYBR^® Premix Ex Taq™ II system (Takara Bio, Inc.). For PCR amplification the following protocol was used: 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec. qPCR was performed in a Bio-Rad IQ5 system (Bio-Rad Laboratories, Inc.). The following primers were used: ATF6, forward 5′-ACAAGACCGAAGATGTCCATTGTG-3′, reverse 5′-GATCCTGGTGTCCATGACCTGA-3′; insulin, forward 5′-CAGCACCTTTGTGGTTCTCACTT-3′, reverse 5′-CTCCACCCAGCTCCAGTTGT-3′; and GAPDH, forward 5′-GGCACAGTCAAGGCTGAGAATG-3′ and reverse 5′-ATGGTGGTGAAGACGCCAGTA-3′.
The amplified products were analyzed by agarose gel electrophoresis (2% agarose) to validate qPCR analysis. Ethidium bromide (cat. no. E1385; Sigma-Aldrich; Merck KGaA) was used for visualization. The standard curve and corresponding values for each sample were determined using the Bio-Rad IQ5 system. GAPDH was used as the internal control, and the 2^−ΔΔCq method was used to calculate the relative expression levels of the target genes (15 (link)).