The Renilla luciferase gene as a reference gene for normalization of gene expression in transiently transfected cells

Background: The importance of appropriate normalization controls in quantitative real-time polymerase chain reaction (qPCR) experiments has become more apparent as the number of biological studies using this methodology has increased. In developing a system to study gene expression from transiently transfected plasmids, it became clear that normalization using chromosomally encoded genes is not ideal, at it does not take into account the transfection efficiency and the significantly lower expression levels of the plasmids. We have developed and validated a normalization method for qPCR using a co-transfected plasmid.Results: The best chromosomal gene for normalization in the presence of the transcriptional activators used in this study, cadmium, dexamethasone, forskolin and phorbol-12-myristate 13-acetate was first identified. qPCR data was analyzed using geNorm, Normfinder and BestKeeper. Each software application was found to rank the normalization controls differently with no clear correlation. Including a co-transfected plasmid encoding the Renilla luciferase gene (Rluc) in this analysis showed that its calculated stability was not as good as the optimised chromosomal genes, most likely as a result of the lower expression levels and transfection variability. Finally, we validated these analyses by testing two chromosomal genes (B2M and ActB) and a co-transfected gene (Rluc) under biological conditions. When analyzing co-transfected plasmids, Rluc normalization gave the smallest errors compared to the chromosomal reference genes.Conclusions: Our data demonstrates that transfected Rluc is the most appropriate normalization reference gene for transient transfection qPCR analysis; it significantly reduces the standard deviation within biological experiments as it takes into account the transfection efficiencies and has easily controllable expression levels. This improves reproducibility, data validity and most importantly, enables accurate interpretation of qPCR data. © 2010 Jiwaji et al; licensee BioMed Central Ltd.

Botched business:the damaging process of reorganising local government 2006-2008, Michael Chisholm and Steve Leach

Book review: Michael Chisholm and Steve Leach, Douglas McLean Publishing, 2008, 175 pp., £ 17.99 (pb), ISBN: 9780946252695

Unique reporter-based sensor platforms to monitor signalling in cells

Introduction - In recent years much progress has been made in the development of tools for systems biology to study the levels of mRNA and protein, and their interactions within cells. However, few multiplexed methodologies are available to study cell signalling directly at the transcription factor level. Methods - Here we describe a sensitive, plasmid-based RNA reporter methodology to study transcription factor activation in mammalian cells, and apply this technology to profiling 60 transcription factors in parallel. The methodology uses two robust and easily accessible detection platforms; quantitative real-time PCR for quantitative analysis and DNA microarrays for parallel, higher throughput analysis. Findings - We test the specificity of the detection platforms with ten inducers and independently validate the transcription factor activation. Conclusions - We report a methodology for the multiplexed study of transcription factor activation in mammalian cells that is direct and not theoretically limited by the number of available reporters.

What do people think about when they answer theory of planned behaviour questionnaires? A 'think aloud' study

Two studies aiming to identify the nature and extent of problems that people have when completing theory of planned behaviour (TPB) questionnaires, using a cognitive interviewing approach are reported. Both studies required participants to 'think aloud' as they completed TPB questionnaires about: (a) increasing physical activity (six general public participants); and (b) binge drinking (13 students). Most people had no identifiable problems with the majority of questions. However, there were problems common to both studies, relating to information retrieval and to participants answering different questions from those intended by researchers. Questions about normative influence were particularly problematic. The standard procedure for developing TPB questionnaires may systematically produce problematic questions. Suggestions are made for improving this procedure. Copyright © 2007 SAGE Publications.

Essentials of Jamaican taxation

This fourth edition of Essentials of Jamaican Taxation as with earlier editions covers the material for an undergraduate course in Jamaican taxation and deals with the theory of taxation and its application through the main provisions of the Income Tax Act, the various Acts governing payroll taxes, the General Consumption Tax Act and the Contractors Levy Act. This edition covers changes which took place after 2007 including the main provisions in the 2012 budget. The text is designed to give mainly students of accounting a working knowledge of taxation as it is administered in Jamaica so they are equipped to perform computations of tax payable by businesses/employers, employees, self-employed, and persons with income from sources other than employment. Most of the text should be understood quite readily by readers other than students who wish to be informed on taxation matters.