Methods in molecular biology
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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.
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DNA cytosine methylation is one of the most abundant epigenetic marks found in the plant nuclear genome. Bisulfite sequencing (BS-Seq) is the method of choice for profiling DNA cytosine methylation genome-wide at a single nucleotide resolution. ⋯ By deep sequencing of the bisulfite converted genomic DNA, the methylation level of each mappable cytosine position in the genome could be measured. In this chapter, we present a detailed 2-day protocol for performing a BS-Seq experiment and a simple bioinformatic workflow for wet lab biologists to visualize the methylation data.
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Whole genome bisulfite sequencing (WGBS) enables the detection of DNA methylation at single base-pair resolution. The treatment of DNA with sodium bisulfite allows the discrimination of methylated and unmethylated cytosines, but the power of this technology can be limited by the input amounts of DNA and the length of DNA fragments due to DNA damage caused by the desulfonation process. ⋯ Briefly, genomic DNA is sheared, end-repaired, 3'-adenylated, and ligated to adaptors with fewer cleanup steps in between, minimizing DNA loss. The adapter-ligated DNA is then treated with sodium bisulfite and amplified with few PCR cycles to reach the yield needed for sequencing.
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Esophagogastrectomy for esophageal adenocarcinoma remains a procedure of significant morbidity and mortality rates. Management should be within the context of a multidisciplinary team. ⋯ Enhanced recovery after surgery (ERAS) protocols are evidence based and improve outcome. Standard protocol for open transthoracic esophagectomy is described.
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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.