OVL2 and SHL2 H9c2 cells were incubated with or without 10 µM ABA , BT-11 , and AR-42 for 4 h after being serum-starved for 18 h. Total RNA was extracted from cardiomyocytes using RNeasy Micro Kit (Qiagen, Milan, Italy), according to the manufacturer’s instructions. cDNA was obtained from 1 μg of total RNA by using iScript cDNA Synthesis Kit (Bio-Rad, Milan, Italy) and qPCR reactions were performed in an iQ5 Real-Time PCR detection system (Bio-Rad, Milan, Italy) as described in [4 (link)]. Specific rat primers were designed using Beacon Designer 2.0 software (Bio-Rad, Milan, Italy), and their sequences are listed in Table S7 .
Values were normalized on hypoxanthine-guanine phosphoribosyltransferase-1 (Hprt1) mRNA expression. Statistical analysis of the qPCR was performed using the iQ5 Optical System Software version 1.0 [Bio-Rad Laboratories, Milan, Italy] by 2−△△Ct method [8 (link)]. The dissociation curve for each cycle of amplification was analyzed to confirm the absence of nonspecific PCR products.
Values were normalized on hypoxanthine-guanine phosphoribosyltransferase-1 (Hprt1) mRNA expression. Statistical analysis of the qPCR was performed using the iQ5 Optical System Software version 1.0 [Bio-Rad Laboratories, Milan, Italy] by 2−△△Ct method [8 (link)]. The dissociation curve for each cycle of amplification was analyzed to confirm the absence of nonspecific PCR products.
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