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Latest issue / 13 December 2011 / Mirco Castoldi. Department of Pediatric Hematology, Oncology and Immunology University of Heidelberg
Cell-free nucleic acids circulating in human blood were first described in 19481. However, it was not until the work of Sorengon and colleagues was published in 19942 that the importance of circulating nucleic acid (cfNA) was recognised. Today, the detection of diverse type of cfNA3 in blood and other body fluids is a valuable resource for the identification of a novel biomarker4,5. Although different types of cfNA have been described (including DNA, mRNA and microRNA), this review focuses on the isolation, detection and clinical utility of circulating microRNAs.
microRNAs (miRNAs) are an abundant class of short single stranded non-coding RNAs (~22 nts) that regulate gene expression at the posttranscriptional level. Interaction between an miRNA and any given of its mRNA targets results in either translation inhibition, mRNA degradation or a combination of both mechanisms. Therefore, miRNAs activity effectively reduces the transcriptional output of a target gene, without affecting its transcription rate. Currently, the sequence of over 60,000 microRNAs are deposited in the miRBase database [Version 17, April 20116]. miRNA activity has been associated with the control of a wide range of basic processes such as development, differentiation and metabolism. Detection of differential expression of miRNAs in many cases have established the basis for miRNA functional analysis and specific miRNA expression patterns can provide valuable diagnostic and prognostic indications, for example, in the context of human malignancies7,8. Moreover, the deregulation of the expression of miRNAs has been shown to contribute to cancer development through various kinds of mechanisms, including deletions, amplification or mutations involving miRNA loci, epigenetic silencing, as well as the dysregulation of transcription factors that target specific miRNAs9,10. (more…)
Featured news, News / 26 October 2010 / The Scott Partnership Ltd
Thermo Fisher Scientific Inc., the world leader in serving science, today announced that it is sponsoring an informative webinar, “The Future of qPCR: Best Practices, Standardization and the MIQE Guidelines.” The webinar, hosted by Science/AAAS, will feature an expert panel discussing the important role qPCR plays in research and molecular diagnostics labs, as well as some of the technical challenges associated with it. Most of these limitations can be addressed with standardized best practices, many of which are outlined in the recently published Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. The webinar will take place on September 30, 2010, and registration is available by following this link www.sciencemag.org/webinar/.
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Featured news, News / 12 October 2010 / Bio-Rad
Bio-Rad’s recently introduced SsoFast™ probes supermix is a reagent that enables researchers using fluorogenic probes to enhance the speed, reliability, and sensitivity of their qPCR experiments.
By combining Bio-Rad’s patented Sso7d fusion polymerase with an optimized buffer formulation, the SsoFast probes supermix yields fast duplex qPCR results in 30 minutes (more…)
Issue 3 2010, Past issues / 24 June 2010 / Ehsan G. Karimiani, Stephan Mohr & Philip J. R. Day,
University of Manchester
Cancer molecular pathology broadly relies on the comparison between diseased and normal tissues, with statistically validated differences revealing cancerassociated pathways. This approach, although comparatively one-dimensional, has been remarkably successful, enabling identification of many types of malignant biomarkers and providing the means to develop pharmaceutical agents directed against pertinent biological targets. Most typically during the progression of malignancies, pathologists employ morphological screening of cancerous tissues. However, this form of monitoring has significant limitations, particularly in the early stages of pre-treatment or during the clinical remission. (more…)
Issue 2 2010 / 9 May 2010 / European Pharmaceutical Review
A diverse and widely applicable laboratory technique, qPCR is vital for the progression of drug discovery, enabling detection and quantification and commonly used for both diagnostic and basic research. This roundtable brings together experts from a wide range of pharmaceutical applications to discuss current technologies and future applications of qPCR for drug discovery and the pharmaceutical industry. (more…)
Issue 1 2010 / 22 February 2010 /
Date 7 – 9 April 2010
Venue Juridicum, University Vienna
Website http://www.qpcr2010-vienna.net
The ongoing evolution of qPCR
The focus of this years qPCR 2010 event will be ‘The ongoing evolution of qPCR’ representing all new and emerging techniques, applications and data analysis methods. HRM, microRNA, CNV, single-cell qPCR, digital PCR, and analysis of circulating nucleic acids will be in the focus of the conference.
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Issue 1 2010 / 22 February 2010 /
Disorders of the immune systems leading to chronic inflammation and allergies are increasing in modern societies. While the possible causative factors are both environmental and nutritional, prevention and even curative options may be derived from our diet. Because background levels of cytokine expression in the general population are generally low, this model was developed to mimic an acute pro-inflammatory threat by a bacterial lipopolysaccharide (LPS).
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Issue 6 2009, Past issues / 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…)
Issue 5 2009, Past issues / 9 October 2009 /
A pivotal attraction of qPCR technology is its apparent lack of complication; an assay consisting of the simple procedure of combining oligonucleotides, PCR mastermix buffer and nucleic acid template to produce a qPCR reaction is perceived as undemanding. This practical simplicity is complemented by the absence of any requirement for post-assay handling, as well as the development of user-friendly data analysis software that makes data generation and visualisation in the shape of amplification plots remarkably simple. However, as we have set out in the first four articles of this series, the translation of an attractive amplification plot into accurate and meaningful data is far from trivial and requires a range of additional considerations. (more…)
Issue 4 2009, Past issues / 30 July 2009 /
The deceptive simplicity of a typical qPCR assay is an important reason for the exponential growth in the adoption of qPCR-related technologies for both research and diagnostic applications. The only requirements for obtaining ostensibly quantitative data are a mixing of primers, DNA and a mastermix, their distribution into individual tubes or wells, turning on a qPCR instrument and collection of threshold cycles (Cqs). Indeed, it is remarkably difficult to make a reaction fail completely but alarmingly simple to produce poor quality data1. Furthermore, it is of great concern that many ready-to-run commercially available systems adopt protocols that discourage the user from performing assay optimisation or validation steps, resulting in the publication of vast volumes of potentially meaningless data. Inevitably this has lead to inaccurate conclusions and publication retractions2,3. Consequently, assay optimisation, validation and critical data evaluation are essential practice if the integrity of the scientific study is to be preserved4. (more…)
Issue 3 2009, Past issues / 29 May 2009 /
Real-time PCR (qPCR) data are reliable only if they result from a robust qPCR assay that has been carefully designed, validated and optimised. This process requires an extensive assay design procedure aimed at generating an optimum primer/probe/amplicon combination to allow accurate quantification of nucleic acids with minimum need for post-PCR analyses (see Figure 1). (more…)
Issue 2 2009, Past issues / 20 March 2009 /
The fluorescence-based quantitative real-time polymerase chain reaction (qPCR)1-3, is the most widely used method to detect and measure minute amounts of DNA in a wide range of samples extracted from numerous sources. Since all currently available thermostable polymerases are DNA-dependent, RNA must be converted (“reverse transcribed”) into DNA prior to its amplification reaction. Both qPCR and reverse transcription (RT)-qPCR have revolutionised life sciences, agriculture, medical research and diagnostic and forensic applications4,5.
However, the adoption of protocols combining RT and qPCR necessitates acceptance of assumptions carried through from legacy RT-PCR; primarily that the process of RT is reproducibly quantitative, linear and maintains proportionality between genes when the cDNA for each is produced. It has become apparent that each of these assumptions requires further qualification before total adoption of any protocol is appropriate. In addition, the degree to which different protocols, RT enzymes and priming strategies influence quantitative assessments also requires clarification. (more…)
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