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.
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.
Real-time PCR has rapidly become the preferred technique for quantitative analysis of nucleic acids. Its superior sensitivity, reproducibility and dynamic range make it the preferred choice for expression profiling in scientific, as well as routine, applications.
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. In this review we discuss the findings on the role of chemokines, as obtained from animal studies. We will focus on the quantification of chemokines and chemokine receptors, making use of the innovative real-time quantitative PCR technique.
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.
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 biomarkers1-3. An important first step for further functional characterisation is the information about differential miRNA expression in cellular processes such as; differentiation4,5, proliferation or apoptosis,6 that may determine which disease causing genes are specifically regulated by miRNAs, or vice versa; which genes regulate miRNA expression.
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.
Biomarkers (biological markers) have become an integral part of both drug discovery and drug development and play an important role in the transition of potential new drugs from discovery into clinical drug development. In the past, most biomarkers were proteins/peptides and metabolites measured by technologies such as immunoassays, enzymatic assays, HPLC and mass spectrometry.
The real-time PCR technique is one of the emerging techniques that, although only described for the first time about a decade ago, have become the method of choice for quantification of DNA and RNA levels in cells, tissues and tissue biopsies.
Quantitative real-time PCR, the workhorse of any genomics lab, is a well established technique that has numerous uses due to its simplicity and flexibility. In this article we will review a brief history of real time PCR, discuss our strategy for optimising a lab running Quantitative-RT-PCR and describe how this technique fits into the drug discovery process. We will conclude with some on-going issues regarding RT-PCR data generation and analysis.
The expansion of microarray-based gene expression studies has led to an increase in demand for gene-specific PCR-based methods for independent validation of results. Although a number of technologies are available to meet this requirement the most popular is currently real-time PCR.
