Dna engine pcr instrument

Total RNA in heart tissue was isolated using Trizol reagent (Invitrogen Life Technologies) according to the manufacturer's protocol. Concentrations of total RNA were analysed by measuring UV light absorbance at 260 nm with a spectrophotometer (ND-100; NanoDrop Technologies). Complementary DNA was synthesised using the SuperScript_III First-Strand Synthesis for RT-PCR kit (cat no. 18080-051; Invitrogen) with DNA Engine PCR instrument (Bio-RAD). Quantitative PCR was performed in triplicate on an Abi Prism 7500 apparatus (Applied Biosystems) according to optimised PCR protocols (15) . The primers for MnSOD (forward, 5′-CACTCTTCCTGACCTGCCTTAC-3′; reverse, 5′-TAGACGTCCCTGCTCCTTATTA-3′) and reference gene β-actin (forward, 5′-CAGCTACGTTGGTGATGAAGCC-3′; reverse, 5′-CAAGAAAGATGGCTGGAAGAGG-3′) were used for the amplification reactions, respectively. We used the relative standard curve method to quantify gene expression, as previously described (15) . The results are expressed as the ratio of MnSOD mRNA abundance:β-actin mRNA abundance.

The concentrations of total RNA were estimated by measuring ultraviolet light absorbance at 260 nm with a spectrophotometer (NanoDrop 2000; Gene Company Ltd). In brief, complementary DNA was synthesised using the SuperScript_III First-Strand Synthesis for RT-PCR kit (Invitrogen) with DNA Engine PCR instrument (Bio-Rad). The FAS and ME mRNA expression levels were determined by relative quantitative realtime PCR using the power SYBR ® Green PCR mater mix kit (catalogue no. 4367659; Applied Biosystems) with ABI Prism 7500 (Applied Biosystems) as described by Li et al. (29) . The information of primers was listed in Table 1. The β-actin was used as the internal reference to normalise the FAS and ME mRNA expression levels using the 2 ÀΔΔCt method (30, 31) . All of the samples were measured in duplicate.