qPCR - Articles and news items

Article 2: Reverse Transcription – a necessary evil

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 applications_4,5.

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…)

The Data Cave: A collaborative method for interpreting genomic data

Issue 2 2009, Past issues / 20 March 2009 /

Generating knowledge and insight from complex genomic data sets is always a challenging endeavor. As data collection becomes more routine and less expensive, and the existing body of data expands, getting the most out of genomics experiments requires ever more expertise and insight. Here, we discuss our method of integrating gene expression profiling data for a candidate oncology drug with pre-existing data and the knowledge and expertise of a wide variety of biologists and statisticians.

A current genomics lab often has access to a wide variety of genomics techniques, including expression profiling via qRT-PCR, mRNA microarrays, and miRNA microarrays; gene knockdown via siRNA and miRNA mimics; a number of pathway tools, and statistical/visualisation programs. In addition, public sources of data and years of accumulation of in-house data allow additional insight. Incorporating these types of data is now standard in drug discovery programs for target identification, mechanism of action studies, toxicity analysis and biomarker discovery. (more…)

Article 1: the importance of sample quality for qPCR

Issue 1 2009, Past issues / 7 February 2009 /

The fluorescence-based quantitative real-time polymerase chain reaction (qPCR)_1-3, has the ability to detect and measure minute amounts of DNA in a wide range of samples extracted from numerous sources. In combination with reverse transcription (RT), the use of this technology has revolutionised life sciences, agriculture and medical research_4,5. In addition, many diagnostic applications have been developed, including microbial quantification, cancer recurrence risk assessment, gene dosage determination, identification of transgenes in genetically modified foods, and detection of extremely low copy targets for forensic investigations_6-11. The simplicity of assay design and execution, together with sensitivity and specificity have made this the method of choice for nucleic acid quantification that is reflected by the prodigious number of peer-reviewed publications reporting qPCR data. Inevitably, a corresponding number of different protocols, reagent recipes, analysis methods and reporting formats are also being used, which is resulting in the scientific literature being corrupted with publications reporting insignificant and conflicting results. The inexperienced or casual qPCR user is vulnerable to the production of inaccurate data because even assays of extremely poor quality usually yield results that are amenable to statistical manipulation. (more…)

Real-time polymerase chain reaction – towards a more reliable, accurate and relevant assay

Issue 6 2008, Past issues / 3 December 2008 /

The fluorescence-based quantitative real-time polymerase chain reaction (qPCR)_1-3 has become firmly established as the preferred technology for the detection and quantification of nucleic acids in molecular diagnostics, life sciences, agriculture and medicine_4,5.

The combination of conceptual and practical simplicity, large dynamic range of linear quantification, speed, sensitivity and specificity has made it the yardstick for nucleic acid quantification not just in basic research, but has engendered numerous uses ranging from basic research through diagnostic and forensic application to treatment monitoring in a clinical setting_6-11. (more…)

There is more to qPCR than the PCR reaction

Issue 5 2008, Past issues / 29 September 2008 /

The polymerase chain reaction is arguably the most significant technical discovery yet to have been made in the field of molecular biology and genetics, if not all life science. It cannot be overstated how much of an impact this technique has had, resulting in molecular biology becoming an integral part of most biological and medical research fields. PCR is both highly versatile and extremely sensitive, yet with basic training and adherence to key precautions against contamination, it is comparatively simple to perform. However, when conducting PCR, there are a number of additional considerations that are frequently overlooked when investigating the presence or amount of nucleic acid within a sample.

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Real-time quantitative PCR – opportunities and pitfalls

Issue 4 2008, Past issues / 2 August 2008 /

The emergence of next generation sequencing technology has brought the prospect of digital analyses closer, technology that will allow not just the quantification of nucleic acids, but will result in the fine-tuning of this information with respect to tissue- and cell-specific transcription, the identification of new transcriptional units, e.g. the detection of new splice variants and their overall correlation with genomic elements. Until that time, the real-time quantitative polymerase chain reaction (qPCR) continues as the enabling technology par excellence offering an unrivalled combination of simplicity, cost-efficiency, accuracy and availability, with application in every area of life sciences and medicine1. Its sensitivity, specificity, and wide linear dynamic range makes qPCR today’s method of choice for any research and diagnostic application that aims to detect and measure nucleic acids2.

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Analysis of microRNA expression by qPCR

Issue 6 2007, Past issues / 23 November 2007 /

Alteration of microRNA (miRNA) expression in a disease compared to a healthy state and/or correlation of miRNA expression with clinical parameters (like disease progression or therapy response), may indicate that miRNAs can serve as clinically relevant biomarkers_1-3. An important first step for further functional characterisation is the information about differential miRNA expression in cellular processes such as; differentiation_4,5, proliferation or apoptosis₆, that may determine which disease causing genes are specifically regulated by miRNAs, or vice versa; which genes regulate miRNA expression.

Whatever the question you would like to address, the precise information about the level of miRNA expression in a specific cell type or tissue is often considered an important first step. A range of methods can be used for the isolation and profiling of miRNAs. Two recent reviews on microRNA₇ and qPCR₈ in European Pharmaceutical Review addressed both topics individually in great detail, but not their combination. This article aims to provide an insight into the application of quantitative real-time PCR (qPCR) to assay microRNA expression. (more…)

The real-time reverse transcription polymerase chain reaction – treat with caution

Issue 5 2007, Past issues / 21 September 2007 / Stephen A. Bustin, Academic Department of Surgery, Institute of Cell and Molecular Science, Queen Mary’s School of Medicine and Dentistry, University of London

The real-time reverse transcription polymerase chain reaction (RT-qPCR) has become the enabling technology par excellence in every field of molecular research and development, including that of clinical drug development and discovery. Its ability to detect as well as quantify RNA biomarkers sensitively, specifically and speedily has made it an indispensable tool in translating the identification of complex biological processes into an understanding of their roles in disease pathology, response to therapy and associated pharmacological proof-of-concept drug efficacy and toxicity studies.

The principal advantages of the real-time polymerase chain reaction (qPCR) are its capacity to generate quantitative data over a wide dynamic range, coupled with high through-put, speed and convenience1, along with its potential for generating more reliable data2. The addition of a reverse transcription (RT) step3 has extended these benefits to the quantification of messenger, regulatory and genomic RNAs, making RT-qPCR to-day’s de facto standard for RNA analysis4,5, even in resource-limited settings6. RT-qPCR assays combine exquisite sensitivity and specificity; turn-around time from sample receipt to result can be less than two hours (the details of the assay5 and appropriate protocols7,8 are described elsewhere). They are performed in a closed system, thus minimising the risk of contamination and quantitative results are calculated using the threshold cycle (Ct), which is the cycle fraction when the real-time PCR instrument first detects the fluorescence generated during a successful amplification reaction. The Ct is obtained during the exponential part of the amplification process, when the most accurate measurement of accumulation is possible. The higher the starting copy number of the nucleic acid target, the sooner a significant increase in fluorescence is observed. Real-time monitoring permits not only accurate quantification of target copy numbers but also critical assessment of the reaction itself. The extensive choice of chemistries, enzymes and instrument platforms available for RT-qPCR is another reason for its immense appeal, albeit at the risk of substantial potential for the generation of discordant results9. (more…)