Quantitative Analysis of HSP72 mRNA

Total RNA was extracted from bEnd.3 cells by using TRIzol reagent (Invitrogen, CA, USA) and converted to cDNA using MMLV reverse transcriptase (Takara, Tokyo, Japan). For real-time PCR, SYBR qPCR Real-Time kit (Takara, Tokyo, Japan) was used according to the manufacturer’s instructions and amplified with the real-time PCR detection system (Bio-Rad). Amplification conditions were set as 40-cycle program (95°C for 15 s, 60°C for 30 s, 72°C for 45 s) (Zhou et al., 2017b (link)). The mRNA level of HSP72 gene was normalized to β-actin, and the results were analyzed using 2^−△△CT method as described previously (Wang et al., 2017 (link)). The PCR primers to detect HSP72 mRNA used in qRT-PCR were 5′-GTGCGTGGGCGTGTTCC-3′ and 5′-CGGTGTTCTGCGGGTTCA-3′, respectively. The PCR primers used to detect β-actin were 5′-CAGCCACCCGAGATTGAGCA-3′ and 5′-TAGTAGCGACGGGCGGTGTG-3′, respectively.