Issue 6 2009

Article 6: qPCR data analysis 2 – Controls and Troubleshooting

Issue 6 2009 / 12 December 2009 /

The tremendous increase in the number of laboratories using qPCR and publications relying on qPCR data are testament to the rapid uptake of this technology. When preceded by reverse transcription (RT-qPCR) it is regarded as the reference technique for validation of previously derived data such as from microarray studies and as the output with which to measure transcript changes after pathway disruption such as by transfection with siRNA or shRNA.

The rapid adoption of the technology is, in part, due to the simplicity with which data are derived. An RT-qPCR experiment requires the combination of PCR primers, cDNA template and DNA polymerase in a suitable buffer, as for legacy PCR but with the addition of either a DNA binding dye such as SYBR Green I dye, or a fluorescent labelled oligo positioned between the two primers (see previous articles in this series [1-5] for details of assay design and choice of detection chemistry). For such an apparently simple technique there are a substantial number of books, articles and conferences discussing the minutiae. It is remarkably difficult to cause the assay to fail completely, making the generation of amplification plots and Cq values almost inevitable. The challenge is to evaluate the data and ensure it represents the original biological question. (more…)

Tagged with: qPCR, Sigma-Aldrich, Stephen Bustin, Tania Nolan

Process Analytical Technology (PAT) in Freeze Drying: Tunable Diode Laser Absorption Spectroscopy as an evolving tool for Cycle Monitoring

Issue 6 2009 / 12 December 2009 /

The most important critical product parameter during a freeze-drying process is the product temperature at the ice sublimation interface, Tp₁. Once the product temperature in this area of interest exceeds the critical formulation temperature (typically denoted as “collapse temperature”, Tc) during primary drying, a stepwise loss of the cake structure may be observed_2,3. This, in turn, can greatly impact the product quality attributes with regard to product appearance, reconstitution times, sub-visible particles and residual moisture content₄. (more…)

Tagged with: Freeze Drying, Henning Gieseler, PAT, Stefan Schneid, University of Erlangen-Nuremberg

IFPAC 2010

Issue 6 2009 / 12 December 2009 /

IFPAC is a meeting place for the latest developments in Process Analytical Technology (PAT) and Quality by Design (QbD). The Twenty-Fourth International Forum and Exhibition will be held at the Baltimore Marriott Waterfront on 31 January – 4 February 2010 www.ifpac2010.org. (more…)

Tagged with: IFPAC, Show Preview

Basics of image analysis in High Content Screening

Issue 6 2009 / 12 December 2009 /

Automated high content screening platforms are capable of producing thousands of images per day. The challenge is to use appropriate analysis methods to extract the maximum amount of biologically-relevant information from these images. In this article we summarise the basic concepts of image analysis and highlight examples of both open-source and commercial software that are available for use with image data sets generated using high-throughput methods. (more…)

Tagged with: HCS (High Content Screening), Jeremy C. Simpson, University College Dublin, Vasanth Singan

The Original HCA Event

Issue 6 2009 / 12 December 2009 /

Coverage Includes: Compound/siRNA Screening – Pathway Analysis – Data Management – Image Analysis – HCA for Stem Cells – Live-Cell Imaging – Flow Cytometry – Neuronal Screening – New Biological Models for HCA – Novel Probes and Biosensors. (more…)

Tagged with: HCA (High Content Analysis), Show Preview

RNAi screening in the era of high-throughput genetics

Issue 6 2009 / 12 December 2009 /

The use of RNAi screening to identify potential drug targets has enjoyed great success in recent years as a robust method for linking genes to a disease process through a functional assessment of a gene in an experimental model1. True, RNAi screening is complicated by problems such as off-target effects and toxicities associated with various properties of RNAi molecules, but effort from many groups has produced a coherent set of guidelines that are useful for most RNAi screens2-4. As such, large scale RNAi screens can be performed by moderately-resourced laboratories. RNAi screens have identified genes involved in resistance to anticancer chemotherapeutics, viral and bacterial infection and specific signal transduction pathways. Now that the technical complications have been identified and largely mitigated, RNAi screening has joined other genomic technologies, such as transcriptional profiling, as a method for broadly surveying the human genome for roles in a given disease or a response to a treatment. (more…)

Tagged with: Pfizer, RNAi, Steven Haney

Drug Metabolism and Pharmacokinetics – an overview

Issue 6 2009 / 12 December 2009 /

The journey from molecular target and early drug lead to the clinic is an arduous one with many hurdles to cross prior to developing a successful clinical candidate. The high rate of attrition of drug molecules has forced drug researchers to pay greater attention to drug metabolism and pharmacokinetics (DMPK) of lead molecules at even the earliest stages of drug discovery. Throughout the development of a successful molecule the researcher must bear in mind three important questions: will enough drug reach the target (pharmacokinetics)? What form will it arrive in (metabolism)? And what will it do when it gets there (pharmacodynamics)? These are the main questions that the DMPK scientist attempts to answer. (more…)

Tagged with: DMPK, Jason Lyle

Combining perspectives: Multiscale integration of Stem Cell research

Issue 6 2009 / 12 December 2009 /

The promise of stem cell-based therapy is predicated on harnessing the plasticity of stem cell phenotypes to repair or replace damaged tissues. As technologies for detecting, isolating, modifying, and tracking stem cells improve, the very definition of what constitutes a stem cell is now an open question. Addressing this fundamental problem has triggered an explosion of activity that spans the entire breadth of biological fields, from molecular biology to population biology. While this has clearly increased the gross amount of information concerning stem cells, its net impact is limited by a lack of integrative multiscale models that are readily accessible to researchers from many disciplines. The field of mesenchymal stem cell biology is a good example of the strengths and limitations of this segregative approach. The goal of this brief review is to highlight some of the most promising recent advances in mesenchymal stem cell research, with an emphasis on how data gathered from one level can benefit research across multiple scales. (more…)

Tagged with: Amanda Lund, George Plopper, Integrative Biosciences Institute (IBI), Rensselaer Polytechnic Institute, Stem Cells

Stem cells and regenerative medicine world congress 2010

Issue 6 2009 / 12 December 2009 /

The 4th annual Stem Cells World Congress and exhibition will be held in South San Francisco, the Birthplace of Biotechnology. This year there are two parallel tracks focused specifically on: (more…)

Tagged with: Show Preview, Stem Cells

A sensitive and selective vibrational spectroscopy technique in life sciences

Issue 6 2009 / 12 December 2009 /

At present, the relevance of Raman Spectroscopy (RS) as an analytical tool in pharmaceutical sciences is increasingly obvious. RS is a mode of vibrational spectroscopy based on inelastic scattering of laser light and, like infrared spectroscopy (IR), provides detailed molecular structure information (see Figure 1A). However, contrary to IR, it is applicable to aqueous samples and furthermore sample handling is minimal. It can thus provide information on compounds in their “natural” environment. To illustrate its applicability, RS can be used to study materials in situ, such as pharmaceutical tablets inside polymer containers₁. It is also applied as a process analyser or a process analytical tool₂. Because of its fast and non-invasive character, it is also very suitable for the fast identification of illicit or counterfeit drugs_3,4. (more…)

Tagged with: Cees Gooijer, Freek Ariese, Raman Spectroscopy, Vrije Universiteit

Lab Automation Roundtable

Issue 6 2009 / 12 December 2009 /

Dr Gordon R Alton, Dr Scott Bowes, Dr Sheraz Gul and Chris Molloy discuss Lab Automation in this European Pharmaceutical Review roundtable discussion. (more…)

Tagged with: Altonyx Consulting, Chris Molloy, European ScreeningPort, Gordon R Alton, IDBS, Lab Automation, Novartis Pharma AG, Scott Bowes, Sheraz Gul

LabAutomation2010 – Where Science, Technology and Industry Come Together

Issue 6 2009 / 12 December 2009 /

In 2010 a diverse group of scientists, engineers and professionals from around the world will come together once again for this laboratory automation event. At LabAutomation2010 participants will develop long-lasting connections through networking and expand their minds by exploring cutting-edge information and strategies. (more…)

Tagged with: Lab Automation, Show Preview