Methods in molecular biology
-
The diversity and huge omics data take biology and biomedicine research and application into a big data era, just like that popular in human society a decade ago. They are opening a new challenge from horizontal data ensemble (e.g., the similar types of data collected from different labs or companies) to vertical data ensemble (e.g., the different types of data collected for a group of person with match information), which requires the integrative analysis in biology and biomedicine and also asks for emergent development of data integration to address the great changes from previous population-guided to newly individual-guided investigations. Data integration is an effective concept to solve the complex problem or understand the complicate system. ⋯ Current integration approaches on biological data have two modes: one is "bottom-up integration" mode with follow-up manual integration, and the other one is "top-down integration" mode with follow-up in silico integration. This paper will firstly summarize the combinatory analysis approaches to give candidate protocol on biological experiment design for effectively integrative study on genomics and then survey the data fusion approaches to give helpful instruction on computational model development for biological significance detection, which have also provided newly data resources and analysis tools to support the precision medicine dependent on the big biomedical data. Finally, the problems and future directions are highlighted for integrative analysis of omics big data.
-
Mitochondria are considered the main source of reactive oxygen species (ROS) in the cell. For this reason they have been recognized as a source of various pathological conditions as well as aging. Chronic increase in the rate of ROS production is responsible for the accumulation of ROS-associated damages in DNA, proteins, and lipids and may result in progressive cell dysfunctions and, in a consequence, apoptosis, increasing the overall probability of an organism's pathological conditions. ⋯ In the chapter we describe a relation between mitochondrial membrane potential and the rate of ROS formation. We present different methods applicable for isolated mitochondria or intact cells. We also present experiments demonstrating that a magnitude and a direction (increase or decrease) of a change in mitochondrial ROS production depend on the metabolic state of this organelle.
-
Prospective or "de novo" biobanking is becoming increasingly popular. Biobanks are installed to provide large collections of biological materials for future medical research. ⋯ Therefore, it is vital that all samples are collected and processed in a similar manner according to standardized procedures to ensure high-quality samples and reduce variability in the analytical process. We describe the processes of the centralized biobanking facility at the Leiden University Medical Center (LUMC).
-
Genetic reporter systems provide a good alternative to monitor cellular functions in vitro and in vivo and are contributing immensely in experimental research. Reporters like fluorescence and bioluminescence genes, which support optical measurements, provide exquisite sensitivity to the assay systems. In recent years several activatable strategies have been developed, which can relay specialized molecular functions from inside the cells. ⋯ In recent years, the applicability of BRET has been greatly enhanced by the adaptation of the assay to multiple detection devices such as a luminescence plate reader, a bioluminescence microscope and a small animal optical imaging platform. Apart from quantitative measurement studies of PPIs and protein dimerization, molecular spectral imaging has expanded the scope for fast screening of pharmacological compounds that modulate PPIs by unifying in vitro, live cell and in vivo animal/plant measurement, all using one assay. Using examples from the literature, we will describe methods to perform in vitro and in vivo BRET imaging experiments and some of its applications.
-
Superoxide and hydrogen peroxide produced by mitochondria play important roles in various physiological and pathological processes. This chapter describes a plate-based method to measure rates of superoxide and/or hydrogen peroxide production at specific sites in isolated mitochondria.