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assay. PCR cycles that generate a linear fit with a slope approximately −3.32 (EPCR = [1–10−(1/slope)] × 100). PCR efficiency between 95 and 105 % are considered acceptable. The linearity is denoted by the Rsq value (correlation coefficient), which should be very close to 1 (>0.985). Another quality indicator of your assay is that there should be a difference of approximately 3.3 in CT values between two standard curve points with a tenfold dilution. 23. Most PCR products will melt somewhere in the range of 80–90 °C, although this melting point can vary with the size and sequence of your specific target. Ideally, the experimental samples should yield a single sharp peak within this temperature range, and the melting temperature should be the same for all the samples. Furthermore, both water and −RT should not generate significant fluorescent signal. If the dissociation curve reveals a series of peaks, it indicates that there is not enough discrimination between specific and nonspecific reaction products (for example, due to dimerization of primers), which would render optimization of the qRT-PCR necessary.
Acknowledgement A.M.B. is supported through the Ramón y Cajal program (RYC-2010-06473). A.N.D.C. was supported by CIHR and by University of Glasgow. References 1. Berg AH, Scherer PE (2005) Adipose tissue, inflammation, and cardiovascular disease. Circ Res 96:939–949 2. Ouchi N, Parker JL, Lugus JJ, Walsh K (2011) Adipokines in inflammation and metabolic disease. Nat Rev Immunol 11:85–97 3. Nguyen Dinh Cat A, Jaisser F (2012) Extrarenal effects of aldosterone. Curr Opin Nephrol Hypertens 21:147–156 4. Aghamohammadzadeh R, Heagerty AM (2012) Obesity-related hypertension: epidemiology, pathophysiology, treatments, and the contribution of perivascular adipose tissue. Ann Med 44:S74–S84 5. Rodbel M (1964) Metabolism of isolated fat cells. Effects of hormones on glucose metabolism and lypolisis. J Biol Chem 239: 375–380 6. Zhang HH, Kumar S, Barnett AH, Eggo MC (2000) Ceiling culture of mature human adi-
pocytes: use in studies of adipocyte functions. J Endocrinol 164:119–128 7. Fernyhough ME, Vierck JL, Hausman GJ, Mir PS, Okine EK, Dodson MV (2004) Primary adipocyte culture: adipocyte purification methods may lead to a new understanding of adipose tissue growth and development. Cytotechnology 46:163–172 8. Van Harmelen V, Skurk T, Hauner H (2005) Primary culture and differentiation of human adipocyte precursor cells. Human Cell Cult Protoc 107:125–135 9. Carswell KA, Lee MJ, Fried SK (2011) Culture of isolated human adipocytes and isolated adipose tissue. Methods Mol Biol 806:203–214 10. Armani A, Mammi C, Marzolla V, Calanchini M, Antelmi A, Rosano GM, Fabbri A, Caprio M (2010) Cellular models for understanding adipogenesis, adipose dysfunction, and obesity. J Cell Biochem 110:564–572
