Abi7900ht machine

Total RNA from cells and tissues was extracted using TRIzol reagents (Invitrogen, Carlsbad, CA, USA). RNA was reverse transcribed into cDNA using the Reverse Transcription System Kit (Promega). The reaction mixture was as follows: SYBR Premix Ex Taq II (Bio-Rad, Hercules, CA, USA), 2 μl of cDNA, 5 μl of 2× master mix, 0.5 μl forward/reverse primer and 2 μl of pure water. RT-PCR was performed on an ABI7900HT machine (Applied Biosystems, Foster City, CA) with 3 replicates. Amplification conditions were 95°C for 5 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Primers were obtained from GeneScript (Nanjing, China), and their sequences were as follows: HIF-1α: Forward 5′-TTGCTCATCAGTTGCCACTTCC-3′, Reverse 5′-AGCAATTCATCTGTGCTTTCATGTC-3′; CAIX: Forward 5′-GGATCTACCTACTGTTGAGGCT-3′, Reverse 5′-CATAGCGCCAATGACTCTGGT-3′; GLUT1: Forward 5′-GATTGGCTCCTTCTCTGTGG-3′, Reverse 5′-TCAAAGGACTTGCCCAGTTT-3′; HK2: Forward 5′-GAGCCACCACTCACCCTACT-3′, Reverse 5′-CCAGGCATTCGGCAATGTG-3′; LDHA: Forward 5′-AAGCGGTTGCAATCTGGATTCAG-3′, Reverse 5′-GGTGAACTCCCAGCCTTTCC-3′; and GAPDH: Forward 5′-TGACGTGGACATCCGCAAAG-3′, Reverse 5′-CTGGAAGGTGGACAGCGAGG-3′. Data were calculated by the 2^-ΔΔCq method after normalization to β-actin mRNA level.

Real-time PCR was used to detect the mRNA expression levels of LC3, the transcription factor EB (TFEB), the sterol regulatory element binding protein 1 (SREBP1C), the sterol regulatory element binding protein-2 (SREBP2), peroxisome proliferator-activated receptor-alpha (PPARα) and peroxisome proliferator-activated receptor γ (PPARγ), the key genes to regulate lipid metabolism in liver. The sequences are listed in Table 2. Briefly, total RNA from the liver was extracted by TRIZOL according to the manufacturer instructions then quantified by UV spectrophotometry. Total RNA samples with the value of A260/280 between 1.8 and 2.0 were used. Reverse transcription reaction (RT) of total RNA in each sample was performed according to the manufacturer’s instructions. The qRT-PCR was carried out in triplicate for target mRNAs of each sample by qPCR SYBR Green mix and specific oligo primers with the following parameters: 1 cycle, 95°, 5 s; 40 cycles, 95°C 10s; 57°C, 30 s. The fluorescent signals of SYBR Green for each amplicon were detected by ABI 7900HT machine (Applied Biosystems, Forster, CA, USA). Melting curve analyses was used to assess the specificity of the product. The 2^-ΔΔCT method was adopted for the data analysis and β-actin was as reference.

The mRNA expression levels of LC3, p62/SQSTM1, Beclin-1 and TFEB in aorta tissues and PPARα, PPARγ, SREBP1C and FAS in liver tissues were detected by quantitative real-time PCR. The primer sequences are given in Table 1. Brifly, total RNA from aorta and liver tissues was extracted by Trizol, and 1 μg of RNA was used to generate cDNA using a Primerscript RT Reagent Kit (Takara Bio). Reverse transcription reactions (RT) were performed according to the manufacturer’s instructions. The qRT-PCR analysis was performed in triplicate for target mRNAs using a qPCR SYBR Green Mix kit (Takara Bio), and the PCR conditions were as follows: 1 cycle of 95°C, 5 s; 40 cycles of 95°C, 10 s; 57°C, 30 s. The fluorescent signals of SYBR Green were subjected to cDNA analysis using an ABI 7900HT machine (Applied Biosystems, Forster, CA, USA). Melting curve analysis was performed to confirm specificity. The 2^-ΔΔCT method was used for the semi-quantitative PCR analysis. Target RNA levels were normalized to β-actin mRNA.

Total RNA was isolated from frozen cell pellets using the RNeasy mini kit (Qiagen Valencia, CA, USA), including the on-column DNase digestion according to the manufacturer’s protocol, and quantitated using a NanoDrop spectrophotometer. The Superscript III kit (Invitrogen) was used to synthesize cDNA from total RNA (0.25 μg) using oligo-dT primers according to the manufacturer’s protocol. FAM TaqMan gene expression probes hs00751002_s1 (CK2α), hs00176505_m1 (CK2α′), hs00414449_m1 (CDK11), and hs01003267_m1 (HPRT-1), TaqMan Fast 2X Mastermix and UNG amperase were from Applied Biosystems, Inc. (Foster City, CA, USA). Reactions were run according to manufacturer’s specification using 96-well FAST plates on an ABI 7900HT machine (Applied Biosystems, Inc.). Analyses were performed using the SDS 2.3 ABI software (Applied Biosystems, Inc.) and changes calculated according to the 2^{(−∆∆Ct)} method. HPRT-1 was used as the reference gene for normalization. All results are reported as the average of reactions run in duplicate.

Amplifications of 50 ng cDNA were performed with an ABI7900HT machine (Applied Biosystems, Carlsbad, CA, USA) in 10 μl reaction mixtures containing 1 × TaqMan Universal PCR Master Mix (Applied Biosystems), 200 nM of primers and 0.25 μl of dual-labeled probe (Roche ProbeLibrary, Roche Applied Science, Basel, Switzerland). The reaction parameters were as follows: 2 minutes 50°C hold, 30 minutes 60°C hold and 5 minutes 95°C hold, followed by 45 cycles of 20 s 94°C melt and 1 minute 60°C anneal/extension. Primer sequences and Roche Library Probe numbers were as follows: CCL2, forward primer 5′-catccacgtgttggctca-3′, reverse primer 5′-gatcatcttgctggtgaatgagt-3′, probe #62; IL6, forward primer 5′-gatggatgctaccaaactggat-3′, reverse primer 5′-ccaggtagctatggtactccaga-3′, probe #6; iNOS, forward primer 5′-ctttgccacggacgagac-3′, reverse primer 5′- tcattgtactctgagggctga-3′, probe #13. Measurements were performed in triplicates and results were analyzed with an ABI sequence detector software version 2.3 using the ΔΔCt method for relative quantification.