Methods in molecular biology
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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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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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Pathological assessment of tissue is the gold standard for diagnosis and staging of neoplasia and provides key prognostic information for clinical management. Proper macroscopic assessment and cut-up technique is essential to ensure that the overall assessment is correct and reproducible. Endoscopic mucosal resection is a technique used for removing early neoplastic glandular lesions of the esophagus at the level of submucosa. Here, we describe the macroscopic assessment and dissection techniques used for the routine handling of endoscopic mucosal resection specimens in the clinical laboratory.
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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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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.