Relative abundance of chemerin mRNA transcript and protein in pituitaries of pigs during the estrous cycle and early pregnancy and associations with LH and FSH secretion during the estrous cycle

The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data. Copyright 2001 Elsevier Science (USA).

Obesity is an alarming primary health problem and is an independent risk factor for type II diabetes, cardiovascular diseases, and hypertension. Although the pathologic mechanisms linking obesity with these co-morbidities are most likely multifactorial, increasing evidence indicates that altered secretion of adipose-derived signaling molecules (adipokines; e.g. adiponectin, leptin, and tumor necrosis factor alpha) and local inflammatory responses are contributing factors. Chemerin (RARRES2 or TIG2) is a recently discovered chemoattractant protein that serves as a ligand for the G protein-coupled receptor CMKLR1 (ChemR23 or DEZ) and has a role in adaptive and innate immunity. Here we show an unexpected, high level expression of chemerin and its cognate receptor CMKLR1 in mouse and human adipocytes. Cultured 3T3-L1 adipocytes secrete chemerin protein, which triggers CMKLR1 signaling in adipocytes and other cell types and stimulates chemotaxis of CMKLR1-expressing cells. Adenoviral small hairpin RNA targeted knockdown of chemerin or CMKLR1 expression impairs differentiation of 3T3-L1 cells into adipocytes, reduces the expression of adipocyte genes involved in glucose and lipid homeostasis, and alters metabolic functions in mature adipocytes. We conclude that chemerin is a novel adipose-derived signaling molecule that regulates adipogenesis and adipocyte metabolism.

We have determined the relationship between mRNA and protein expression levels for selected genes expressed in the yeastSaccharomyces cerevisiaegrowing at mid-log phase. The proteins contained in total yeast cell lysate were separated by high-resolution two-dimensional (2D) gel electrophoresis. Over 150 protein spots were excised and identified by capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS). Protein spots were quantified by metabolic labeling and scintillation counting. Corresponding mRNA levels were calculated from serial analysis of gene expression (SAGE) frequency tables (V. E. Velculescu, L. Zhang, W. Zhou, J. Vogelstein, M. A. Basrai, D. E. Bassett, Jr., P. Hieter, B. Vogelstein, and K. W. Kinzler, Cell 88:243–251, 1997). We found that the correlation between mRNA and protein levels was insufficient to predict protein expression levels from quantitative mRNA data. Indeed, for some genes, while the mRNA levels were of the same value the protein levels varied by more than 20-fold. Conversely, invariant steady-state levels of certain proteins were observed with respective mRNA transcript levels that varied by as much as 30-fold. Another interesting observation is that codon bias is not a predictor of either protein or mRNA levels. Our results clearly delineate the technical boundaries of current approaches for quantitative analysis of protein expression and reveal that simple deduction from mRNA transcript analysis is insufficient.