When it comes to DNA research, scientists often find themselves weighing digital PCR (dPCR) against the more established quantitative real-time PCR (qPCR). Which technique is better to use? The answer is both.
During the last 30 years exceedingly sensitive and accurate technologies have been developed to measure and quantify amounts of nucleic acids and proteins. With techniques such as quantitative real-time PCR (qPCR) and nextgeneration sequencing (NGS) DNA, messenger RNAs (mRNA), micro RNAs (miRNA), long non-coding RNAs (lncRNA) and today even proteins can be measured in most types of samples allowing the comparison of environmental conditions, studies of diseases and monitoring of treatments.
Although it is still early days in terms of our understanding of epigenetics, the fast development of new tools and technologies to define genome-wide epigenetic variations in humans has the potential to enable effective new epigenetic therapies and diagnostic tests for a wide range of diseases beyond and including cancer.
Quantitative real-time PCR (qPCR) has during the last two decades emerged as the preferred technology for nucleic acid analysis in routine as well as in research. qPCR has the sensitivity to detect a single molecule, the specificity to differentiate targets by a single nucleotide, and, because of its exponential nature, virtually unlimited dynamic range1.
The recent availability of tailored automation solutions that combine liquid handling at the reading position with the latest innovations in low light imaging technology are providing screeners with new tools for the measurement of high throughput flash luminescence.These systems are proving particularly suited to the study of GPCR activation, using photoproteins such as aequorin as indicators of intracellular calcium.This article reviews the advantages of aequorin flash luminescence, compares the latest detection instrumentation, discusses the availability of new aequorin-based assay services and highlights the development of several new and improved photoproteins that have potential in the detection of calcium release triggered by several receptors.
QPCR instruments are able to monitor amplicon quantity in real-time during PCR reactions by detecting fluorescence signals and recording fluorescence data.
Employing fast QPCR cycling protocols is a simple and effective way of maximising throughput by reducing run durations. Fast cycling protocols can easily be achieved by reducing the temperature step dwell times.
DNA contamination can often occur in quantitative reverse transcription – polymerase chain reactions (QRT-PCR), and should be removed in order to avoid false positive results.
G protein-coupled cell surface receptors (GPCRs) have served a fundamental role in modern pharmacology, due to their central importance in cell communication, and have been the target for the discovery of a large number of drugs. By one estimate, more than 40% of marketed drugs target GPCRs.
This review provides an insight into real-time quantitative PCR (qPCR) assays today.
Quantitative real-time PCR is becoming mature technology for the quantification of nucleic acids. It is spreading wide outside its original use in the research laboratories, becoming preferred technology for a range of applications, many that require specialised solutions and adaptations. Integration with pre-analytical steps and post-processing operations are becoming key challenges.