Methods in molecular biology
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Traumatic brain injury (TBI) diagnoses have increased in frequency during the past decade, becoming a silent epidemic. The pathophysiology of TBI involves pathophysiological processes affecting the brain, induced by traumatic biomechanical forces resulting in temporary impairment of neurological function. Preclinical models have been generated to recapitulate the mechanical, neuroinflammatory, and behavioral outcomes observed in the clinical setting. ⋯ The model is reproducible and can be adjusted to produce a mild to moderate and severe injury, as reflected by mortality and return of reflexes, by adjusting the amount of force applied. The histopathological changes achieved with this model reproduce that seen in human TBI including focal contusion in the cortex, with accompanying intraparenchymal punctate hemorrhage, followed by inflammation and neuronal degeneration. This chapter describes the LFP model, which produces a mixed model of focal and diffuse brain injury that progresses over time affecting predominantly the cortical parenchyma.
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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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Aberrations of the DNA methylome contribute to onset and progression of diseases. Whole genome bisulfite sequencing (WGBS) is the only analytical method covering the complete methylome. ⋯ In tagmentation-based WGBS (TWGBS), several DNA and time-consuming steps of the conventional WGBS library preparation are circumvented by the use of a hyperactive transposase, which simultaneously fragments DNA and appends sequencing adapters. TWGBS requires only nanogram amounts of DNA and, thus, is well suited to study precious biological specimens such as sorted cells or micro-dissected tissue samples.
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Esophageal adenocarcinoma is heterogeneous and studies have reviewed many important mutations that contribute to the pathogenesis of the cancer. These discoveries have helped paved the way into identifying new gene markers or gene targets to develop novel molecular directed therapy for better patient outcomes in esophageal adenocarcinoma. Despite the recent bloom in next-generation sequencing, Sanger sequencing still represents the gold standard method for the study of the driver genes in esophageal adenocarcinoma. This chapter focuses on the sequencing techniques in identification of single gene mutations.
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Single-cell transcriptome sequencing, often referred to as single-cell RNA sequencing (scRNA-seq), is used to measure gene expression at the single-cell level and provides a higher resolution of cellular differences than bulk RNA-seq. With more detailed and accurate information, scRNA-seq will greatly promote the understanding of cell functions, disease progression, and treatment response. ⋯ Particularly, we present a protocol to discover and validate cancer stem cells (CSCs) using scRNA-seq. Suggestions have also been made to help researchers rationally design their scRNA-seq experiments and data analysis in their future studies.