Sybr green real time pcr master mix 2

TG (10522) and PBA (P21005) were purchased from Cayman Chemicals (Ann Arbor, MI, USA) and Sigma-Aldrich (Saint Louis, MO, USA), respectively. Western blot antibodies, including BiP (3183), CHOP (5554), Caspase-3 (9662), Bax (2772), TNFα (3707), p.JNK (4671), T.JNK (9252), p.P38 (9211), T. P38 (8690), p.IRS1-ser307 (2381), T.IRS (2382), β-Actin (4970), and secondary HRP conjugated (7074), were purchased from Cell Signaling Technology (Danvers, MA, USA), and Bcl-2 (ab59348) was obtained from Abcam (Waltham, MA, USA). The Amersham ECL select (RPN2235) reagent kit, Halt™ Protease and Phosphatase Inhibitor Cocktail (78440) and PVDF membrane (IPVH00010) were purchased from GE Health Care and Millipore, respectively. PCR materials were YTzol (Yekta Tajhiz Azma, Tehran, Iran, #YT9065), total RNA isolation kit (RiboEX GeneAll, Seoul, South Korea), 301-902), cDNA Synthesis Kit (Yekta Tajhiz Azma, Tehran, Iran, #YT4500) and SYBR Green Real-Time PCR Master Mix (2×) (Ampliqon, Copenhagen, Denmark).

Total RNA was isolated from the hippocampus using YTzol according to the manufacturer’s recommendations. The purity and concentration of total RNA were measured by a spectrophotometer NanoDrop 2000 (Thermo Scientific, Wilmington, DE, USA). cDNA synthesis was carried out using 500 ng of total RNA and oligo(dT) primer according to the kit instructions, incubated at 42 °C for 60 min, and 70 °C for 5 min enzyme inactivation.
To quantify the expression of ER stress marker genes (s-XBP1/us-XBP1, ATF4, ATF6), the qPCR reaction was done using SYBR Green Real-Time PCR Master Mix (2×) (Ampliqon, Copenhagen, Denmark) on an ABI System (Applied Biosystems, Carlsbad, CA, USA). Primers are listed in Table 1. Reaction cycling conditions were 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. In the present study, β-actin was employed as the housekeeping gene, and all genes were normalized to β-actin level using the 2^−ΔΔCt method.