Quantitative real-time PCR for studies of microbial gene expression

现代微生物学，就像许多生命科学一样，需要应用分子方法来解决基因多样性、丰富性和调控问题。聚合酶链式反应(PCR)是分子微生物学工具箱中的日常必需品，因为它允许扩增单个基因靶标，用于序列分析、克隆、核酸杂交和许多其他应用。在过去的几年里，仪器已经很容易用于实时聚合酶链式反应，其中一个人可以测量在聚合酶链式反应过程中单基因扩增的速度，以便定量样本中特定模板分子的原始数量。定量实时聚合酶链式反应(QPCR)正迅速成为一项不可或缺的技术，甚至正在取代Northern印迹杂交等分子生物学经典方法来量化基因表达(一旦RNA分子转化为cDNA)。我的工作包括比较多种氨氧化细菌在高和低氧分压下以及在暴露于亚硝化剂、亚硝酸盐和一氧化氮时的整体基因表达(通过基因芯片杂交)，以寻找与亚硝化应激反应和一氧化二氮产生有关的基因。一旦确定了这些基因，就会仔细检查它们在野生型和突变背景下的调节和生理功能，并使用相同的基因来检测它们在自然生态系统样本中的表达水平。所有这些活动都需要qPCR来：1)验证来自微阵列杂交的全基因组表达数据，2)确定突变细菌与野生型细菌或暴露于不同刺激(例如亚硝酸盐、低氧、一氧化氮)的细菌中的基因表达的相对水平，以及3)确定从环境样品中提取的DNA或RNA混合物中的基因模板的原始数量。由于其检测下限低，能够扩增模板的任何部分，不仅允许查询基因的直接表达，还允许查询RNA的处理，以及通过使用多个荧光团在单个PCR反应中检测多个模板的能力，qPCR是非常强大的。

Modern day microbiology, like many life sciences, necessitates the application of molecular methods to address questions of gene diversity, abundance, and regulation. The polymerase chain reaction (PCR) is an everyday staple of the molecular microbiology tool chest as it allows for the amplification of individual gene targets for sequence analysis, cloning, nucleic acid hybridization, and many other applications. In the past few years, instrumentation has become readily available for real-time polymerase chain reaction, wherein one can measure the rate of single gene amplification during the PCR reaction in order to quantify the original number of a specific template molecule in a sample. Quantitative real-time PCR (qPCR) is rapidly becoming an indispensable technique and is even replacing such molecular biology classics as Northern blot hybridization for quantifying gene expression (once RNA molecules are converted into cDNA). My work involves comparing global gene expression (via cDNA array hybridization) in multiple species of ammonia-oxidizing bacteria under high and low oxygen tension and upon exposure to the nitrosating agents, nitrite and nitric oxide, to find genes involved in nitrosative stress response and nitrous oxide production. Once the genes are identified, their regulation and physiological function in wild-type and mutant backgrounds are carefully examined, and the same genes are used to examine levels of their expression in samples from natural ecosystems. All of these activities require qPCR to: 1) verify whole-genome expression data derived from microarray hybridization, 2) determine relative levels of gene expression in mutated versus wild-type bacteria, or bacteria exposed to different stimuli (e.g. nitrite, low oxygen, nitric oxide), and 3) determine the original amount of a gene template in a mixture of DNA or RNA extracted from environmental samples. qPCR is extremely powerful due to its low detection limit, the ability to amplify any part of a template, allowing one to query not just direct expression of a gene, but also the processing of RNA, and the ability to detect multiple templates in a single PCR reaction by using multiple fluorophores.