Freshly harvested heart tissue sections (10 mg) were snap-frozen in liquid nitrogen and homogenized in TRIzol reagent (Invitrogen, Carlsbad, CA; weight/volume ratio, 1:10). Freshly cultured cells (106/well, 6-well plates) were directly homogenized in TRIzol reagent. Total RNA was extracted by chloroform/isopropanol/ethanol method, treated with RNase free DNase I (NEB, Beverly MA), and assessed for quality (OD260/280 ratio ? 2.0) and quantity (OD260 of 1 = 40 ?g/ml RNA). Total RNA (2 ?g) was reverse transcribed by using poly (dT)18 oligonucleotide with an iScript kit (Bio-Rad, Hercules CA). The cDNA was utilized as a template, and quantitative real-time PCR was performed on an iCycler Thermal Cycler with SYBR-Green supermix (Bio-Rad) and gene-specific oligonucleotide pairs. The PCR Base Line Subtracted Curve Fit mode was applied for threshold cycle (Ct), Ct values for target mRNAs were normalized to GAPDH mRNA, and the relative expression level of each target gene was calculated as 2??Ct (?Ct = Ct sample—Ct control) . The oligonucleotide pairs used for amplifying the mRNAs are listed in Table A in S1 Table.
All animal experiments were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and were approved by the Institutional Animal Care and Use Committee at the University of Texas Medical Branch, Galveston (protocol number: 805029).