7900ht fast rt qpcr system

RNA from cell lines was harvested using a miRNA isolation kit (mirVana, Ambion, Inc., 2130 Woodward Street, Austin, TX, USA) for miRNA and a TRIzol procedure for mRNA. The concentration and quality of the extracted RNA were determined by measuring OD₂₆₀ and the OD₂₆₀:OD₂₈₀ ratio. First, 150 ng RNA were reverse transcribed to cDNA with specific stem-loop RT primers using a TaqMan microRNA reverse transcription kit (Applied Biosystems) for miRNA, and then 150 ng RNA were reverse transcribed to cDNA with random primers using a high-capacity cDNA reverse transcription kit (Applied Biosystems) from TaqMan for mRNA samples. RT-qPCR was performed using an ABI 7900HT fast RT-qPCR system and a TaqMan universal master mix (Applied Biosystems). All the primers were obtained from the TaqMan miRNA and mRNA assay kits (Applied Biosystems). The endogenous microRNA RNU43 [58 (link)] was used as an internal control for miRNA expression, and the housekeeping gene GAPDH was used as an internal control for mRNA expression.

Total RNA was extracted from RAW264.7 macrophages and the PBMCs of gout patients and healthy controls patients using TRI-zol reagent (TaKaRa, Japan). Reverse transcription was performed using the PrimeScript® RT reagent kit. The cDNAs of miR-223 and U6 were synthesized using stem-loop reverse transcription primers (miR-223: 5′-GTC​GTA​TCC​AGT​GCA​GGG​TCC​GAG​GTA​TTC​GCA​CTG​GAT​ACG​ACT​GGG​GT-3′; U6: 5′-AAC​GCT​TCA​CGA​ATT​TGC​GT-3′). Following pre-amplification, gene expression was assessed on a 7900HT Fast RT-qPCR System (Applied Biosystems), using the manufacturer’s recommended protocol. SYBR Green II was used to compare the relative expression levels of specific mRNAs (including IL-1β, NLRP3, ASC, and caspase1 mRNAs). RT-qPCR was performed to obtain a mean CT value for each sample. The CT values of the samples were compared using the 2^−ΔΔCT method, and β-actin expression was used as an internal reference. The primer sequences are shown in Table 1.

RNA was isolated from retroperitoneal adipose tissue and the SVF with TRI Reagent (Sigma-Aldrich, St. Louis, MO, USA). RNA was isolated from the liver and the upper gut (duodenum) using the RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA concentration was determined using the NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE, USA). Reverse transcription was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Warrington, UK). Quantitative real-time polymerase chain reaction (RT-qPCR) analysis was performed on the ABI 7900HT Fast RT-qPCR System using Sequence Detection System 2.4 software to quantify mRNA expression using TaqMan Fast Advanced Master Mix and pre-designed TaqMan Gene Expression Assays (Applied Biosystems, Warrington, UK; Table S1 in Supplementary Material). To control for variability between samples, the relative amounts of the genes were normalized to peptidylprolyl isomerase A (Ppia), hypoxanthine–guanine phosphoribosyltransferase 1 (Hprt1), and/or glyceraldehyde 3-phosphate dehydrogenase (Gapdh) expression. The comparative CT method (2^–ΔΔCT) was used to analyze data (23 (link)).