7500 fast real time

Rat kidney tissues were treated by a homogenizer (UH-48, Union-Biotech, Shanghai, China). Total miRNAs from the obtained homogenate and NRK-52E cells were extracted by TRIzol lysis buffer (15596018, ThermoFisher). First strand cDNAs from the isolated miRNAs were synthesized by Synthesis Kits (K1621, ThermoFisher, USA). QPCR reaction was performed on an Applied Biosystems PCR machine (7500 FAST real-time, Applied Biosystems, Foster City, CA, USA) with the TB Green Premix Ex Taq II (Tli RNaseH Plus) (RR820Q, TAKARA, China). The primers used were shown as follows: miR-155 (Forward: 5′-GGGTGTCGTATCCAGTGCAA-3′; Reverse 5′-GTCGTATCCAGTGCGTGTCG-3′) and U6 (Forward: 5′-CTCGCTTCGGCAGCACA-3′; Reverse: 5′-AACGCTTCACGAATTTGCGT-3′). Amplification conditions were set as follows: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The relative gene expression was normalized to U6 and quantified using the 2^–ΔΔCT method [38 (link)]. This experiment was repeated three times.

The blood was collected from the all subjects using an EDTA-K₂ anticoagulant blood vessel. The DNA was extracted according to the phenol-chloroform method and stored at -80°C. The genotyping was performed in an Applied Biosystems 7500 Fast Real-Time quantitative polymerase chain reaction (qPCR) system with the following TaqMan SNP Genotyping Assays kit (Applied Biosystems) according to the manufacturer's instructions. Each PCR reaction mixture (10 μL) contained DNA template (0.4 μL), ddH₂O (4.35 μL), 2×TaqMan Universal PCR Master Mix (5 μL), and 20× SNP Genotyping Assay Mix (0.25 μL). The genomic DNA was amplified at 95°C for 10 min, followed by 40 cycles of 95°C for 15s and 60°C for 60s. All genotyping reagents and analytical software were purchased from Applied Biosystems. Approximately 5% of the samples were randomly repeated to validate the genotyping procedures, and the concordance rate was 100%. The results of the genotyping were analyzed with 7500 Fast System V1.4.0 SDS software.

To investigate a wide spectrum of genes involved in lipid metabolism the Human Adipogenesis RT² Profiler PCR Array (Qiagen) was used. 84 “metabolic” genes were analyzed in addition to positive control genes (beta actin and Ribosomal protein). Real-time PCR was performed with 7500 Fast Real-Time (Applied Biosystems) using Sybr green detection. Results were analyzed by RT² Profiler PCR Array Data Analysis. Transcriptional levels of genes were shown as fold change vs Healthy Donors, values >1.50 or <0.5 were considered significantly modified. All dataset was submitted in Geo Repository http://www.ncbi.nlm.nih.gov/geo. Accession number: GSE54432