Gene-specific monitoring of T7-based RNA amplification by real-time quantitative PCR.

Contains fulltext : 186317.pdf (publisher's version ) (Open Access)T7-based RNA amplification is applied when there is insufficient RNA. The overall extent of amplification can be measured spectrophotometrically (i.e., quantifying RNA yields), but this measurement does not provide information about the RNA amplification of individual genes. Here we describe a method applying real-time quantitative PCR, which enables the monitoring of RNA amplification of individual genes. The amount of RNA before and after T7-based RNA amplification was determined by real-time quantitative PCR for three housekeeping genes: beta-2-microglobulin, porphobilinogen deaminase, and serine dehydratase, which are, respectively, a high, intermediate/low, and low copy transcript. Real-time quantitative PCR appeared to be suitable to determine the extent of RNA amplification, as was reflected by the low intra- and inter-run coefficients of variation of cycle threshold of 1.1%-2.1%. The application of real-time quantitative PCR showed that T7-based RNA amplification is reproducible but might introduce a sequence-specific bias. Real-time quantitative PCR is a novel approach to monitor RNA amplification and is particularly suited to study RNA amplification of individual genes

A thorough QT study to assess the effects of tbo-filgrastim on cardiac repolarization in healthy subjects

Liat Adar,1 Noa Avisar,1 Andreas Lammerich,2 Robert B Kleiman,3 Ofer Spiegelstein1 1R&D, Teva Pharmaceutical Industries Ltd, Netanya, Israel; 2Biosimilars Clinical Development, CPP Teva ratiopharm, Merckle GmbH, Ulm, Germany; 3Global Cardiology, eResearch Technology Inc, Philadelphia, PA, USA Abstract: Tbo-filgrastim is a recombinant human granulocyte colony-stimulating factor approved by the US Food and Drug Administration to reduce the duration of severe neutropenia in patients with nonmyeloid malignancies receiving myelosuppressive anticancer drugs associated with a clinically significant incidence of febrile neutropenia. We assessed the effect of tbo-filgrastim on cardiac conduction and repolarization in healthy subjects. A three-arm, parallel-group, active- and placebo-controlled, double-blind study randomized healthy adults to a single 5 µg/kg intravenous tbo-filgrastim infusion, a single intravenous placebo infusion, or a single 400 mg moxifloxacin oral dose. The primary end point was placebo-corrected time-matched change from baseline in QT interval corrected using a QT individual correction (QTcI) method. Secondary end points included heart rate, PR interval, QRS duration, change in electrocardiogram patterns, correlation between QTcI change from baseline (milliseconds) and tbo-filgrastim serum concentrations, and safety variables. A total of 145 subjects were enrolled (50 tbo-filgrastim, 50 placebo, 45 moxifloxacin). Peak placebo-corrected change from baseline for QTcI with tbo-filgrastim was 3.5 milliseconds, with a two-sided 95% upper confidence interval of 7.2 milliseconds, demonstrating no signal for any tbo-filgrastim effect on QTc. Concentration-effect modeling showed no evidence of an effect of tbo-filgrastim on cardiac repolarization. Tbo-filgrastim produced no clinically significant changes in other electrocardiogram parameters. Tbo-filgrastim was well tolerated. Keywords: tbo-filgrastim, electrocardiogram, QT interval, granulocyte colony-stimulating factor&nbsp

Pharmacokinetics, pharmacodynamics and safety of CEP-26401, a high-affinity histamine-3 receptor antagonist, following single and multiple dosing in healthy subjects

Stress-related psychiatric disorders across the life spa