Real-time PCR based on SYBR-Green I fluorescence: An alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions

Background

Real-time PCR is increasingly being adopted for RNA quantification and genetic analysis. At present the most popular real-time PCR assay is based on the hybridisation of a dual-labelled probe to the PCR product, and the development of a signal by loss of fluorescence quenching as PCR degrades the probe. Though this so-called 'TaqMan' approach has proved easy to optimise in practice, the dual-labelled probes are relatively expensive.

Results

We have designed a new assay based on SYBR-Green I binding that is quick, reliable, easily optimised and compares well with the published assay. Here we demonstrate its general applicability by measuring copy number in three different genetic contexts; the quantification of a gene rearrangement (T-cell receptor excision circles (TREC) in peripheral blood mononuclear cells); the detection and quantification of GLI, MYC-C and MYC-N gene amplification in cell lines and cancer biopsies; and detection of deletions in the OPA1 gene in dominant optic atrophy.

Conclusion

Our assay has important clinical applications, providing accurate diagnostic results in less time, from less biopsy material and at less cost than assays currently employed such as FISH or Southern blotting.     

Understanding high diastereomeric discrimination in formation of oligoribonucleotide phosphorothioate linkages: the first study of pKa-dependent activation in solid-supported coupling of 2'-O-substituted ribonucleoside phosphoramidites

Activation of 2'-O-substituted ribonucleoside phosphoramidites with various activators during solid-supported synthesis of phosphorothioate oligonucleotides was studied. The Rp:Sp diastereomeric composition of resulting phosphorothioate linkage dependent on pKa of activator utilized for coupling.     

Mitochondrial,sarcoplasmic membrane integrity and protein degradation in heart and skeletal muscle in exercised rats

Several different exercise regimens varied in the severity of tissue damage induced. Therefore, this study investigated the effects of a single bout of exercise versus endurance training in heart and skeletal muscles with different predominant fiber types on indices of mitochondrial, endoplasmic reticulum (ER) integrity and protein degradation. Male Wistar rats performed different treadmill exercise protocols: exhaustive, maximal exhaustive, eccentric, training and exhaustive exercise after training. The maximal and eccentric exercises resulted in a significant loss of integrity of the sarcoplasmic and ER muscle, while no changes were observed in cardiac muscle. Mitochondrial membrane fluidity measured by the fluorescence polarization method was significantly increased post-acute exercises in heart and oxidative muscles. Regular exercise can stabilize and preserve the viscoelastic nature of mitochondrial membranes in both tissues. The highest increase in carbonyl content was obtained in heart after exhaustive exercise protocol, from 1+/-0.1 to 3.6+/-0.14 nmol mg protein(-1), such increase were not found after regular exercise with values significantly decreased. Nitrate heart levels showed attenuated generation of nitric oxide after training. Muscle protein oxidation was produced in all exhaustive exercises including eccentric exercise.     

Ginseng increases intestinal elimination of albendazole sulfoxide in the rat

Herbal products show potential drug interactions, some of them with adverse effects. The main aim of this work was to study the effect of Panax ginseng on the intestinal elimination of the benzimidazole derivative albendazole sulfoxide (ABZSO). An upper small intestine segment was isolated and perfused in situ with saline, while ABZSO solution (10 mg/kg i.v.) was administered intravenously. Blood samples and intestinal secretion were collected over 60 min and analysed by HPLC. The intestinal clearance of ABZSO was 0.106+/-0.010 ml/min. Systemic co-administration of ginseng (10 mg/kg i.v.) increased significantly (P<0.05) the clearance of ABZSO (0.132+/-0.005 ml/min). The increase in ABZSO elimination could be the result of the effect of ginseng on metabolic pathways. These results highlight the interactions between herbal products (sometimes dietary constituents) and drugs such as benzimidazoles, since ginseng modifies the luminal clearance of this anthelminthic drug and could potentially interfere with drugs that undergo the same intestinal processes.     

Maneuvering for advantage: the genetics of mouse susceptibility to virus infection

Genetic studies of host susceptibility to infection contribute to our understanding of an organism's response to pathogens at the immunological, cellular, and molecular levels. In this review we describe how the study of host genetics in mouse models has helped our understanding of host defense mechanisms against viral infection, and how this knowledge can be extended to human infections. We focus especially on the innate mechanisms that function as the host's first line of defense against infection. We also discuss the main issues that confront this field, as well as its future.     

Smoking impairs muscle recovery from exercise

Cigarette smoking is a leading cause of many adverse health consequences. Chronic nicotine exposure leads to insulin resistance and may increase the risk of developing non-insulin-dependent diabetes mellitus in young otherwise healthy smokers. To evaluate smoking-induced effects on carbohydrate metabolism, we studied muscle glycogen recovery from exercise in a young healthy population of smokers. The study used 31P-13C NMR spectroscopy to compare muscle glycogen and glucose 6-phosphate levels during recovery in exercised gastrocnemius muscles of randomized cohorts of healthy male smokers (S) and controls (C). Data for the two groups were as follows: S, > or =20 cigarettes/day (n = 8), 24 +/- 2 yr, 173 +/- 3 cm, 70 +/- 4 kg and age- and weight-matched nonsmoking C (n = 10), 23 +/- 1 yr, 175 +/- 3 cm, 67 +/- 3 kg. Subjects performed single-leg toe raises to deplete glycogen to approximately 20 mmol/l, and glycogen resynthesis was measured during the first 4 h of recovery. Plasma samples were assayed for glucose and insulin at rest and during recovery. Test subjects were recruited from the general community surrounding Yale University. Glycogen was depleted to similar levels in the two groups [23.5 +/- 1.2 (S) and 19.1 +/- 1.3 (C) mmol/l]. During the 1st h of recovery, glycogen synthesis rates were similar [13.8 +/- 1.1 (S) and 15.3 +/- 1.3 (C) mmol x l-1 x h-1]. Between hours 1 and 4, glycogen synthesis was impaired in smokers [0.8 +/- 0.2 (S) and 4.5 +/- 0.5 (C) mmol x l-1 x h-1, P = 0.0002] compared with controls. Glucose 6-phosphate was reduced in smokers during hours 1-4 [0.105 +/- 0.006 (S) and 0.217 +/- 0.019 (C) mmol/l, P = 0.0212]. We conclude that cigarette smoking impairs the insulin-dependent portion of muscle recovery from glycogen-depleting exercise. This impairment likely results from a reduction in glucose uptake.     

Cellular remodeling in heart failure disrupts K(ATP) channel-dependent stress tolerance

ATP-sensitive potassium (K(ATP)) channels are required for maintenance of homeostasis during the metabolically demanding adaptive response to stress. However, in disease, the effect of cellular remodeling on K(ATP) channel behavior and associated tolerance to metabolic insult is unknown. Here, transgenic expression of tumor necrosis factor alpha induced heart failure with typical cardiac structural and energetic alterations. In this paradigm of disease remodeling, K(ATP) channels responded aberrantly to metabolic signals despite intact intrinsic channel properties, implicating defects proximal to the channel. Indeed, cardiomyocytes from failing hearts exhibited mitochondrial and creatine kinase deficits, and thus a reduced potential for metabolic signal generation and transmission. Consequently, K(ATP) channels failed to properly translate cellular distress under metabolic challenge into a protective membrane response. Failing hearts were excessively vulnerable to metabolic insult, demonstrating cardiomyocyte calcium loading and myofibrillar contraction banding, with tolerance improved by K(ATP) channel openers. Thus, disease-induced K(ATP) channel metabolic dysregulation is a contributor to the pathobiology of heart failure, illustrating a mechanism for acquired channelopathy.