Polymerase Chain Reaction (PCR) - Articles and news items

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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Night on Highway 128

Issue 2 2008, Past issues / 19 March 2008 /

Most people in molecular biology today are not old enough to remember pre-PCR. But try to do your job without it and you will see what a difference that simple little technique has made.

‘Polymerase Chain Reaction’ is now a word in Merriam Webster’s Collegiate Dictionary and if you put ‘PCR’ into Google, you get 18,000,000 hits. If you type in ‘PCR song,’ you get a lovely little ditty courtesy of Bio-Rad, which will rattle around in your brain like an insane cat in your garage. Try it.

When I stumbled on PCR in the spring of 1983, I was trying to increase the demand for oligonucleotides, which before automation my laboratory had made by hand. Our new machine from my friend Ron Cook at Biosearch across the San Francisco bay had threatened job stability in the laboratory by doing what had taken us about three weeks to do, in eight hours – and it did it every eight hours, no breaks.

My attempt succeeded. The demand went up by about a million and I didn’t have to fire any of my fellow ‘lab’ workers at Cetus. (more…)

The role of chemokines in type 1 diabetes: as assessed by RT-PCR

Issue 2 2008, Past issues / 19 March 2008 /

Type 1 diabetes is an autoimmune disease, characterised by immune infiltration into the islets of Langerhans, resulting in the destruction of insulin producing b-cells. Over recent years, evidence has been collected on the important role of chemokines in the recruitment of immune cells leading to the pathology of this disease. (more…)

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…)

Cutting edge technologies and their potential role in pharmaceutical microbiology

Issue 6 2007 / 23 November 2007 /

In order to meet the challenges demanded by the requirements of Process Analytical Technology (PAT), the modern microbiological laboratory needs to become more innovative in microbial detection, identification and enumeration. Technology is becoming available that will speed up microbiological analysis, potentially allowing pharmaceutical microbiology tests to get as close as is possible to the concepts of PAT. Following on from the article by Bob Johnson1, this article explores the future technologies in greater detail.

The technical requirements for any rapid microbiology method (RMM) include a combination of the following: significantly reduced time-to-result when compared with conventional microbiological methods; automated, miniaturised and high-throughput technology platforms; increased sensitivity, accuracy, precision and reproducibility; detection of a single, viable micro-organism without the requirement for cellular growth; capable of testing for total counts and specified objectionable simultaneously; enhanced detection of stressed organisms. Business requirements include: significant reduction of testing time to release products more rapidly; lower inventories (raw material, in-process material and finished product); reduction of repeat testing, deviations, OOS investigations and product rejection. Furthermore, the system should be portable and user-friendly. (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…)