Methods in molecular biology
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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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Staging is one of the most important factors that determine the prognosis of the patients with esophageal adenocarcinoma. The current staging protocol includes parameters-extent of tumor (T), lymph node status (N), presence of distant metastasis (M), and grade of the adenocarcinoma (G). In addition, different criteria are used in patients with and without neoadjuvant therapy. The silent aspects of the use of new staging protocol are discussed.
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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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Tissue microarray technology could allow immunohistochemical staining or in situ hybridization on hundreds of different tissue samples simultaneously. It allows faster analysis and considerably reducing costs incurred in staining. ⋯ In the literature, many researches of esophageal adenocarcinoma use tissue microarray to enhance the output. In this chapter, we have a brief overview of tissue microarray technologies, the advantages and disadvantages of tissue microarray, and related troubleshootings.
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Exon-skipping therapy is an emerging approach that uses synthetic DNA-like molecules called antisense oligonucleotides (ASOs) to splice out frame-disrupting parts of mRNA, restore the reading frame, and produce truncated yet functional proteins. Phosphorodiamidate morpholino oligomer (PMO) is one of the safest among therapeutic ASOs for patients and has recently been approved under the accelerated approval program by the US Food and Drug Administration (FDA) as the first ASO-based drug for Duchenne muscular dystrophy (DMD). Multi-exon skipping utilizing ASOs can theoretically treat 80-90% of patients with DMD. Here, we describe the systemic delivery of a cocktail of ASOs to skip exon 51 and exons 45-55 in the mdx52 mouse, an exon 52 deletion model of DMD produced by gene targeting, and the evaluation of their efficacies in vivo.