Properties of targeted preamplification in DNA and cDNA quantification

Objective: Quantification of small molecule numbers often requires preamplification to generate enough copies for accurate downstream enumerations. Here, we studied experimental parameters in targeted preamplification and their effects on downstream quantitative real-time PCR (qPCR). Methods: To evaluate different strategies, we monitored the preamplification reaction in real-time using SYBR Green detection chemistry followed by melting curve analysis. Furthermore, individual targets were evaluated by qPCR. Result: The preamplification reaction performed best when a large number of primer pairs was included in the primer pool. In addition, preamplification efficiency, reproducibility and specificity were found to depend on the number of template molecules present, primer concentration, annealing time and annealing temperature. The amount of nonspecific PCR products could also be reduced about 1000-fold using bovine serum albumin, glycerol and formamide in the preamplification. Conclusion: On the basis of our findings, we provide recommendations how to perform robust and highly accurate targeted preamplification in combination with qPCR or next-generation sequencing

The modulation of glucocorticoid receptor content by 3-O-methyl-D-glucose transport in human mononuclear leukocyte in obesity

Glucocorticoid receptors (GR) and 3-O-methyl-D glucose (3-O-MG) transport were determined in mononuclear leukocytes (MNL) from 11 abdominal obese subjects, 10 pituitary-dependent Cushing's syndrome (Cushing's disease) and 10 healthy controls. Using a whole-cell competitive binding assay and H-3-dexamethasone as tracer, MNL of abdominal obese subjects were found to have 4855+/-1389 sites/cell which was significantly lower (p0.05). These results indicated that, in abdominal obesity, the GR binding capacity in MNL is influenced by the changes in glucose transport. (J. Endocrinol. Invest. 21: 656-661, 1998) (C)1998, Editrice Kurtis

Factors contributing to the separation of endodontic files

Cleaning and shaping of the root canal system is essential for successful endodontic treatment. However, despite improvements in file design and metal alloy, intracanal file separation is still a problematic incident and can occur without any visible signs or permanent deformation. Only a few studies have reported high success rates of fractured file removal using contemporary techniques. Conflicting results have been reported regarding the clinical significance of retaining separated files within root canals. An understanding of the mechanisms of, factors contributing to, file fracture is necessary to reduce the incidence of file separation within root canals. This article reviews the factors that are of utmost importance and in light of these, preventive procedures and measures are suggested