Real-time PCR for measuring gene rxpression

重金属和芳烃受体（AhR）配体的复杂混合物的共污染是一个常见的环境问题，具有多种生物后果，包括出生缺陷，免疫抑制，内分泌干扰和癌症。我的长期目标是：1）了解重金属和AhR配体之间的潜在相互作用，这是在环境中常见的，对AhR调节基因的调节，和2）调查AhR配体/金属混合物对AhR配体介导的致突变性和致癌性的影响。实时PCR方法将用于确定基因表达的变化，以响应共同暴露于重金属和AhR配体。多路复用（通过使用多达4种不同的荧光探针同时测量一种以上的基因转录物）将加快这一过程，减少所需的测定总数和所需的RNA量。低容量PCR反应是至关重要的，特别是当评估小病变时，因为可用的RNA量将非常低。应用生物系统7300实时PCR很好地适应了这些特征。鉴定共暴露于金属/AhR配体后表现出差异表达的基因可以帮助我们鉴定新的基因，并找到负责金属介导效应的异常细胞内或细胞间通路。

Co-contamination with complex mixtures of heavy metals and aryl hydrocarbon receptor (AhR)-ligands is a common environmental problem with multiple biological consequences, including birth defects, immune suppression, endocrine disruption and cancer. My long-term objectives are to: 1) understand the potential interaction between heavy metals and AhR ligands, which are common in the environment, on the regulation of AhR-regulated genes, and 2) investigate the effects of AhR ligand/metal mixtures on AhR ligand-mediated mutagenicity and carcinogenicity. Real-time PCR methodology will be used to determine changes in gene expression in response to co-exposure to heavy metals and AhR ligands. Multiplexing (measuring more than one gene transcript concurrently accomplished by employing up to 4 different fluorescent probes) will expedite the process, reducing the total number of assays necessary and the amount of RNA required. Low volume PCR reactions are critical particularly when small lesions are evaluated because the amount of RNA available will be very low. These features are well accommodated for by the Applied Biosystems 7300 real-time PCR. The identification of genes which show differential expression after co-exposure to metals/AhR ligands could help us to identify novel genes and to find the abnormal intracellular or intercellular pathways that are responsible for the metals-mediated effects.