Fluorescence and absorbance detection of ROS

基因表达的调控和基因组完整性的维持是每个生物体的基础。表达一种蛋白质需要基因激活，当不再需要这种蛋白质时，基因就会被关闭。在胚胎中，这些事件是协调的，并允许幼稚细胞接受新的命运。了解基因调控是很重要的，因为这些基因通常在成年后被开启，导致许多疾病。DNA受到化学修饰和容易出错的复制的影响，导致损伤和基因突变的积累。许多因素也影响基因调控和DNA完整性。例如，活性氧（ROS）是细胞代谢氧气和产生能量的副产物，可以破坏DNA、蛋白质和脂质。细胞有防止活性氧损伤的机制，最近我们了解到细胞利用活性氧启动信号级联，调节有利于宿主的基因表达。因此，在正常条件下，在不同的遗传背景下或在环境压力下测量ROS水平是很重要的，如果我们能将这些水平与基因活性和基因组完整性联系起来，将会对ROS在细胞中的功能有更多的了解。然而，在我们能够做到这一点之前，我们必须能够测量活性氧水平，量化基因的活性，DNA损伤水平和突变频率。为此，开发了检测方法来测量ROS水平、DNA损伤和基因活性。虽然需要不同的协议，但检测和量化的仪器是非常相似的。该方法的核心是两年前由NSERC资助的GloMax多重检测系统。这种光度计具有精确测量光子的能力，其检测到的数量与基因活性的数量成正比。购买荧光模块来测量活性氧水平，并拥有专用的紫外线吸收模块来量化蛋白质表达和过氧化物水平，这无疑将增加我们专用的最先进设备，为我们的两个实验室和几名学员提供一个现代化的工具来解决与广泛的生物学相关的问题。

Regulation of gene expression and maintenance of genome integrity are fundamental to every organism. Gene activation is needed to express a protein and when that protein is no longer required the gene is turned off. In embryos these events are coordinated and allow naive cells to adopt new fate. Understanding gene regulation is important because often these genes are turned on later in the adult, leading to a number of maladies. DNA is subject to chemical modification and error-prone replication leading to an accumulation of damage and genetic mutations. Numerous agents also influence gene regulation and DNA integrity. For instance, Reactive Oxygen Species (ROS), which are by-products of a cell's ability to metabolize oxygen and produce energy, can damage DNA, proteins and lipids. Cells have mechanisms to prevent ROS damage and quite recently we learned that cells use ROS to initiate signaling cascades that regulate gene expression that benefit the host. Measuring ROS levels under normal conditions, in different genetic backgrounds or at times following environmental stress is thus important and if we can relate these levels to gene activity and genome integrity, much will be learned about how ROS function in cells. Before we can do this, however, we must be able to measure ROS levels and quantify the activity of genes, the level of DNA damage and the frequency of mutations. Towards that end, detection methods were developed to measure ROS levels, DNA damage and gene activity. Although different protocols are required, the instrumentation for detection and quantification is quite similar. At the heart of the methodology is our GloMax Multi-Detection System that was funded by NSERC two years ago. This luminometer has the capability to accurately measure photons of light, with the amounts detected proportional to the amount of gene activity. The request to purchase the fluorescence module to measure the level of ROS and having a dedicated UV-absorbance module to quantify protein expression and peroxide levels will certainly add to our dedicated state-of-the-art equipment, providing our two labs and several trainees with a modern tool to address relevant questions pertaining to a broad spectrum of biology.