qPCR - Articles and news items

RNA quality matters

Issue 6 2012 / 18 December 2012 / Mikael Kubista, Jens Björkman, David Svec and Robert Sjöback, TATAA Biocenter

RNA levels can be measured with very high specificity, sensitivity and accuracy with techniques such as real-time quantitative PCR (qPCR), microarray analysis and next generation sequencing. This makes messenger (m) RNAs and potentially microRNAs and other non-coding RNAs popular as biomarkers. But RNA is less stable and more dynamic than DNA, and assays are not always specific for RNA, so can we trust measured expression values?

A biomarker is a biological molecule found in blood, other body fluids or tissues, and is a sign of a normal or abnormal process, or of a condition or disease1. The biomarker may be used to see how well the body responds to a treatment for a disease or condition. Most popular and common molecular biomarkers are DNA, RNA and proteins. While proteins and in particular DNA are quite stable molecules and can be analysed for many properties such as sequence years after being removed from their natural biological environment, RNA molecules are not (Table 1). The extra 2’-hydroxyl group on the ribose in RNA that is absent in DNA is a nucleophile. It confers catalytic activity to ribozymes, but also makes RNA intrinsically unstable. In aqueous solution, RNA spontaneously degrades through self-cleavage catalysed by metal ions such as Mg2+, high (>9) or low (<2) pH, and temperature. EDTA or citrate is therefore typically added to RNA preserving solutions to chelate Mg2+2. Although RNA is more resistant to ultraviolet (UV) irradiation than DNA, it causes several types of damage including photochemical modification, cross – linking and oxidation. (more…)

qPCR: In-depth focus 2012

Issue 5 2012, Supplements / 24 October 2012 / European Pharmaceutical Review

This free to view qPCR in-depth focus is supported by Eurogentec, Affymetrix, Roche, TwistDx and TakaRa:

• Setting the bar
  Mikael Kubista and Ales Tichopad, TATAA Biocenter and the Institute of Biotechnology, CAS, Medical Faculty Pilsen, Charles University of Prague
• Q & A
  Mikael Kubista from the TATAA Biocenter poses five questions for Jay Brock, Senior Manager, Applications and Technical Support, USB® Life Science Reagents from Affymetrix
• Not your grandfathers’ real-time PCR
  Dirk P. Dittmer, Professor of Microbiology and Immunology and Member of Lineberger Comprehensive Cancer Center, University of North Carolina

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resDNASEQ® E. coli Residual DNA Quantitation System

Featured news, News / 1 June 2012 / Life Technologies

Integrated sample preparation and real-time PCR assay for the quantitation of E. coli host cell DNA

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Building robust PCR/qPCR assays

Issue 2 2012 / 26 April 2012 / Linda Starr-Spires, Director, Nucleic Acid Methods Platform, Global Clinical Immunology Department, Sanofi Pasteur

The process of building robust PCR/qPCR assays is a matter of perseverance and consistency. A few questions that should be answered prior to starting development will help make the process more efficient and effective:

1. Does the assay need to simply detect the presence of the target (qualitative), or must it assign a value to the detected target (quantitative)? The development process for a qualitative or quantitative assay, although similar in many respects, ultimately will take different paths
2. In what type of matrix will samples be? Matrix plays an important role in both development and validation of the assay. If the assay is needed for multiple matrices (whole blood, plasma, serum, differing tissue types, etc.), each matrix must be evaluated individually to determine its impact on assay performance
3. Will extraction be required, and by what method? 4) What throughput will be needed?

Answering these questions early in the process will help prevent ‘reworks’ later. (more…)

Expression profiling of circulating miRNAs as a novel non-invasive diagnostic tool

Issue 6 2011 / 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…)

Thermo Fisher Scientific and Science/AAAS Host Webinar on the Future of Quantitative PCR and the Importance of Standardization

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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Bio-Rad’s New SsoFast Probes Supermix Accelerates Gene Expression Analysis

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…)

High-throughput PCR based diagnostics: Linking sample handling to molecular oncology risk groups

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…)

qPCR: Current technologies and future applications

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…)

qPCR 2010 Symposium and Exhibition

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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Ex-vivo LPS stimulation model coupled with quantitative PCR and its multispecies application in immunonutrition

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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Article 6: qPCR data analysis 2 – Controls and Troubleshooting

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…)