Methods in molecular biology
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Comparative Study
Comparison of Mitochondrial Incubation Media for Measurement of Respiration and Hydrogen Peroxide Production.
High-Resolution FluoRespirometry is a well-established and versatile approach to study mitochondrial oxygen uptake amperometrically in combination with measurement of fluorescence signals. One of the most frequently applied fluorescent dyes is Amplex UltraRed for monitoring rates of hydrogen peroxide production. Selection of an appropriate mitochondrial respiration medium is of crucial importance, the primary role of which is to support and preserve optimum mitochondrial function. ⋯ Stability of assay sensitivity over experimental time was highest in MiR05 but slightly less in MiR07. Taken together, MiR05 and Buffer Z yield comparable results on respiration and H2O2 production. Despite the lower sensitivity, MiR05 was selected as the medium of choice for FluoRespirometry due to the highest stability of the sensitivity or calibration constant observed in experiments over periods of up to 2 h.
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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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Bisulfite sequencing (BS-seq) enables the detection of DNA methylation at cytosine residues (5mC) at single-nucleotide resolution. For many applications, a limiting factor of conventional BS-seq protocols is the high amount of DNA required, since the treatment with bisulfite causes severe DNA fragmentation. Here, we describe a post-bisulfite tagging method that accounts for this problem. ⋯ The method can also be used to analyze defined fractions of genomes from limited samples by Reduced Representation Bisulfite Sequencing (RRBS). This involves restriction digestion, gel separation and fragment elution prior to BS-seq library preparation to enrich certain areas of the genome. This reduction of represented genomic regions lowers the sequencing cost considerably while providing an accurate assessment of total genome-wide DNA methylation levels and assessment of DNA methylation in categorical genomic regions.
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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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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.