Sensimix 2 probe kit

Genomic DNA from Lower Silesian participants was obtained from peripheral blood lymphocytes using standard phenol–chloroform extraction and ethanol precipitation. Genotyping of studied polymorphisms was performed using polymerase chain reaction and restriction fragment length polymorphism (PCR–RFLP) method described elsewhere [8 (link), 16 (link)].
The DNA for the Centre of Oncology-Institute (Warsaw) group was extracted using the QIAamp DNA Blood Mini Kit (QIAGEN). Genotyping of selected polymorphisms was done using the TaqMan^® SNP Genotyping Assays (Life Technologies), SensiMix II Probe Kit (Bioline) and the ABI Prism^® 7900HT real-time PCR system.

10⁷ Jurkat T-cells were transfected with 50μg pEFTax or pEF. 5x10⁵ 293T cells were transfected with 2μg of pEFTax or pEF (see Transfections). 48h later, total cellular RNA was isolated from transfected Jurkat or 293T cells (RNA isolation Kit II, Macherey-Nagel, Düren, Germany) and reversely transcribed to cDNA using SuperScript II and random hexamer primers (both Life Technologies GmbH). 200ng of cDNA and SensiMix II Probe Kit (BioLine GmbH, Luckenwalde, Germany) were used according to the manufacturer’s instructions for quantitative real-time RT-PCR (qPCR) in an ABI Prism 7500 Sequence Analyzer (Applied Biosystems, Foster City, CA, USA). Primers and FAM (6-carboxyfluorescein) / TAMRA (tetramethylrhodamine)-labeled probes for detection of β-actin (ACTB) and Tax transcripts have been described before [46 (link)]. A TaqMan Gene Expression Assay (Hs00979631_g1; Applied Biosystems) was used for quantitation of Fascin transcripts. Expression levels were computed by interpolation from standard curves generated from plasmids carrying the respective target sequences and calculation of the mean of triplicated samples. Relative copy numbers (rcn) were determined by normalizing copy numbers on those of ß-actin (ACTB). At least, three independent experiments were performed.

Gene expression levels were measured with a 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA) after converting the extracted RNA to cDNA using a SensiMix II probe kit (Bioline, London, UK). The expression levels in each sample were normalized by Gapdh using the ΔΔCt method, and the relative expression level for the control sample was calculated. The following primer sequences were used and verified by PrimerBank (Harvard, MA) (Table S2).

Plasma glucose samples were analyzed immediately using a YSI 2300 StatPlus Glucose Analyzer system (Yellow Springs, OH). All other samples were collected in EDTA tubes, centrifuged for 10 minutes at 4°C and 3000 rpm, and stored at -80°C until later analysis. Samples were batch analyzed in duplicate to minimize inter-assay variability. Circulating HMW adiponectin, total adiponectin, and insulin (Alpco, Salem, NH) as well as leptin (Millipore, Billerica, MA) was determined using enzyme-linked immunosorbent assay kits. FFAs were analyzed using enzymatic colorimetric assay (Wako Diagnostics, Richmond, VA). RNA was extracted from adipocytes using Trizol extraction. One μg of omental RNA was then treated with deoxyribosnuclease (Invitrogen, Carlsbad, CA) and used to reverse transcribe complementary deoxyribonucleic acid (cDNA) using an iScript cDNA synthesis kit (BioRad, Hercules, CA). To quantify gene expression, cDNA was combined with Taqman primer probes (HPRT1-VIC plus one additional FAM-conjugated primer-probe) and SensiMix™ II Probe Kit (BioLine, London, UK). Semi-quantitative real-time PCR was performed on a CFX96 Real-Time System with an annealing temperature of 60°C for all reactions (BioRad, Hercules, CA). Data were calculated by the ΔΔCt method and expressed in arbitrary units that were normalized to HPRT1 levels.

Before and after tri-differentiation, RNA samples were collected from all cell populations to measure gene expressions of transcription factors and proteins involved in differentiation. RNA was isolated using an RNeasy MicroKit (Qiagen), and cDNA was synthesized using an iScript cDNA Synthesis Kit (Bio-Rad) or Superscript IV Reverse Transcriptase (ThermoFisher). iQ SYBR Green Supermix (Bio-Rad) and SensiMix II Probe Kit (Bioline) was used as detection kits, and samples were analyzed on a CFX96 Touch (Bio-Rad). GAPDH was used as the housekeeping gene throughout analysis. Primer sequences can be found in Supplementary Table S1.

All protocols were approved by the Institutional Review Board at the University of Virginia. Plasma was collected from an equal number of patients exhibiting intermittent claudication or as risk factor (e.g., diabetic, hypertensive, hyperlipidemic, and smoker) controls (n = 25/group). A diagnosis of PAD with intermittent claudication was based on having one of the following: (1) an ABI < 0.9; (2) a previous peripheral vascular intervention; or (3) an abnormal TBI, without any corresponding foot ulcers or resting pain. Total RNA was purified from 200 μL plasma using the miRCURY Biofluids RNA isolation kit (Exiqon, 300112). For reverse transcription (Applied Biosystems, 4366596) and miR-199a-5p detection, TaqMan miRNA assays (Applied Biosystems, 4427975) were used. RT-PCR was performed using the SensiMix II probe kit (Bioline) on an CFX96 detection system (Bio-Rad). Gene expression was normalized to RNU6 (Applied Biosystems, 4427975). Relative expression was determined with the comparative 2^ΔΔCt method and is represented as the log₂ fold change. The log₂ fold change data were normally distributed as determined by a Shapiro-Wilk test was tested for statistical significance using a two-sided Student’s t test.