Methods in molecular biology
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Prospective or "de novo" biobanking is becoming increasingly popular. Biobanks are installed to provide large collections of biological materials for future medical research. ⋯ Therefore, it is vital that all samples are collected and processed in a similar manner according to standardized procedures to ensure high-quality samples and reduce variability in the analytical process. We describe the processes of the centralized biobanking facility at the Leiden University Medical Center (LUMC).
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Liquid biopsy of cancers is an area of increasing interest in medical practice for the surveillance, management, and potential detection of malignant cells, using minimally invasive collection of body fluids. A liquid biopsy is particularly useful for metastatic cancers, which may be difficult to be sampled by core biopsy, due to difficulty of access or an occult location. Access to DNA shed from esophageal adenocarcinoma can enable the detection of mutations confirming the presence of malignant cells or the evolution of clonal lines with altered treatment response profiles. In this chapter, we detail a method for the isolation of cell-free DNA from blood plasma and DNA associated with exosomes in blood from patients with esophageal adenocarcinoma.
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Genetic reporter systems provide a good alternative to monitor cellular functions in vitro and in vivo and are contributing immensely in experimental research. Reporters like fluorescence and bioluminescence genes, which support optical measurements, provide exquisite sensitivity to the assay systems. In recent years several activatable strategies have been developed, which can relay specialized molecular functions from inside the cells. ⋯ In recent years, the applicability of BRET has been greatly enhanced by the adaptation of the assay to multiple detection devices such as a luminescence plate reader, a bioluminescence microscope and a small animal optical imaging platform. Apart from quantitative measurement studies of PPIs and protein dimerization, molecular spectral imaging has expanded the scope for fast screening of pharmacological compounds that modulate PPIs by unifying in vitro, live cell and in vivo animal/plant measurement, all using one assay. Using examples from the literature, we will describe methods to perform in vitro and in vivo BRET imaging experiments and some of its applications.
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Mitochondria are considered the main source of reactive oxygen species (ROS) in the cell. For this reason they have been recognized as a source of various pathological conditions as well as aging. Chronic increase in the rate of ROS production is responsible for the accumulation of ROS-associated damages in DNA, proteins, and lipids and may result in progressive cell dysfunctions and, in a consequence, apoptosis, increasing the overall probability of an organism's pathological conditions. ⋯ In the chapter we describe a relation between mitochondrial membrane potential and the rate of ROS formation. We present different methods applicable for isolated mitochondria or intact cells. We also present experiments demonstrating that a magnitude and a direction (increase or decrease) of a change in mitochondrial ROS production depend on the metabolic state of this organelle.
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Somatic copy-number alterations are commonly found in cancer and play key roles in activating oncogenes and deactivating tumor suppressor genes. Digital polymerase chain reaction is an effective way to detect the changes in copy number. In esophageal adenocarcinoma, detection of somatic copy-number alterations could predict the prognosis of patients as well as the response to therapy. This chapter will review the methods involved in digital polymerase chain reaction for the research or potential clinical applications in esophageal adenocarcinoma.