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qPCR - Articles and news items
Issue 6 2014 / 23 December 2014 / Natalia Meani and Manuela Vecchi
Recognised as one of the major scientific breakthroughs of the 20th century, polymerase chain reaction (PCR) is a quick and simple method to create, in a test tube, millions of copies of a given DNA segment from a complex mixture of genetic material. This method has greatly stimulated biochemical, molecular biology and genetic research and, given its ability to amplify DNA from limited amounts of biologic samples, including fossils, opened the way for new applications in medicine, genetics, biotechnology, forensics and paleobiology…
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.
Setting the bar.
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.
Integrated sample preparation and real-time PCR assay for the quantitation of E. coli host cell DNA…
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:
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
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
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.
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.
Thermo Fisher Scientific and Science/AAAS Host Webinar on the Future of Quantitative PCR and the Importance of Standardization
Thermo Fisher Scientific, announced that it is sponsoring a webinar, “The Future of qPCR…
Bio-Rad introduced SsoFast™ probes supermix which enables researchers using fluorogenic probes to enhance the speed, reliability…
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.
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.
ABB Analytical Measurement ABS Laboratories Analytik Jena AG Axis-Shield Diagnostics Ltd Black Swan Analysis Limited CAMO Software AS Celsis International Cherwell Laboratories CI Precision Cobalt Light Systems Comark Instruments Coulter Partners Dotmatics Limited Enterprise System Partners EUROGENTEC GE Analytical Instruments Hyglos GmbH IONICON Analytik GmbH kbiosystems ltd LabWare MKS Umetrics Portalis Ltd Powder Systems Limited (PSL) RADWAG Reach Separations ReAgent Source BioScience Stratech Scientific Limited Thermo Fisher Scientific Tuttnauer Vaisala Ltd Waters Corporation Xylem Analytics Ytron-Quadro (UK) Limited Yusen Logistics