Methods in molecular biology
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After conducting systematic and quantitative comparisons of different sample preparation techniques regarding their capability to efficiently and reproducibly recover proteins from biopsies, we present here our superior protocol for extracting proteins from low amounts of adipose tissue. Adipose tissue as a matrix in bottom-up proteomics is challenging due to the extremely high lipid content. The lysis buffer utilized contains the detergent sodium deoxycholate, which does not impair the activity of trypsin and therefore enables direct digestion without detergent removal steps. The resulting workflow is time saving, cost efficient, easy to perform, and it can also be applied to other hydrophobic samples.
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DNA methylation is a covalent modification of DNA that plays important roles in processes such as the regulation of gene expression, transcription factor binding, and suppression of transposable elements. The use of whole genome bisulfite sequencing (WGBS) enables the genome-wide identification and quantification of DNA methylation patterns at single-base resolution and is the gold standard for analysis of DNA methylation. Computational analysis of WGBS data can be particularly challenging, as many computationally intensive steps are required. ⋯ Second, DNA methylation levels are estimated at each cytosine position using the aligned sequence reads of the bisulfite treated DNA. Third, regions of differential cytosine methylation between samples can be identified. Finally, these data need to be visualized and interpreted in the context of the biological question at hand.
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Next-generation sequencing refers to the high-throughput DNA sequencing technologies, which are capable of sequencing large numbers of different DNA sequences in a single/parallel reaction. It is a powerful tool to identify inherited and acquired genetic alterations associated with the development of esophageal adenocarcinoma. ⋯ Thus, second-generation sequencing methods can provide a complete picture of the esophageal adenocarcinoma genome by detecting and discovering different type of alterations in the cancer. This would help in diagnostics and will further help in developing personalized medicine in esophageal adenocarcinoma.
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The discovery of the CRISPR-Cas9 system raises hope for the treatment of many genetic disorders. We describe here an approach based on the use of a pair of single guide RNAs to form a hybrid exon that does not only restore the dystrophin gene reading frame but also results in the production of a dystrophin protein with an adequate structure of the central rod-domain, with a correct spectrin-like repeat. The therapeutic approach described here involved DMD patient cells having a deletion of exons 51-53 of the DMD gene.
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The aberrant DNA methylation has been noted to occur at promoter of tumor suppressor, cell adhesion, DNA repair, and other growth regulating genes during the progression of nonneoplastic esophageal mucosa to Barrett esophagus to esophageal adenocarcinoma. Methylation-mediated silencing of individual gene or concurrent loss of a number of genes plays crucial roles in dysplasia-metaplasia-neoplasia sequence of esophageal adenocarcinoma. ⋯ There are a number of methods including bead array, PCR and sequencing, pyrosequencing, methylation-specific PCR, and PCR with high-resolution melt curve available to determine the methylation status of particular gene of interest. Herein, we describe the polymerase chain reaction followed by sequencing-based protocol for identifying DNA methylation status in esophageal adenocarcinoma.