Cfx96 sequence detection system

One hundred nanograms of total RNA was reverse-transcribed (RT) and amplified using the iScript One-Step RT-PCR kit for probes (Bio-Rad, Hercules, CA). Real-time qPCR was performed with the Bio-Rad CFX96 sequence detection system using predesigned primer/probe sets against CPT1a, CPT2, and SLC25a20 from Applied Biosystems (Foster City, CA). The relative fluorescence signal was normalized to PPIB using the ddCT method (Livak and Schmittgen, 2001 (link)).

One-hundred nanograms of the total RNA were reverse-transcribed and amplified according to the manufacturer’s instructions for the iScript One-Step RT-PCR kit for probes (Biorad; Hercules, CA). Quantitative real-time PCR (qPCR) was performed with the Biorad CFX96 sequence detection system using commercially available primer/probe sets (Applied Biosystems; Foster City, CA). Nascent NR3C1 RNA was measured with primer/probes spanning either intron 1 (forward: 5′-GCAAATAACTCAGTACAAATGGTCT-3′; reverse: 5′-ACTATTTAACCAAACACCCAAAGG-3′) or the exon 2/intron 2 junction (forward: 5′-CGTGTGGAAGCTGTAAAGTC-3′; reverse: 5′-TGCACCTGAACTAATGTCTATCA-3′). The relative fluorescent signal of the gene of interest was normalized to PPIB using the 2^−ΔΔCT method.

Total RNA of the lungs was extracted using a TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa, Dalian, China), according to the manufacturer's instructions, and quantified with a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE). Complementary DNA was generated with a specific RT primer. RT-PCR was performed with SYBR Premix Ex TaqTM (Tli RNaseH plus) (TaKaRa, Dalian, China) by the Applied Bio-Rad CFX96 Sequence Detection System (Applied Biosystems). The expression level of GPX1-4, CAT, SOD1, SOD2, and UCP2 was defined from the threshold cycle (Ct), and relative expression levels were calculated using the 2^−ΔΔCt method after normalization regarding the expression of U6 small nuclear RNA. The expression level of GAPDH in the tissues was taken as the negative control. Three independent assays were performed. The information of the primers is shown in Table 1.