Ultralow freezer for gene expression analysis

用于基因表达分析的超低温冰箱

• 批准号: 359975-2008
• 负责人: Marrone, DianoFabio
• 金额: $ 0.86万
• 依托单位: Wilfrid Laurier University
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments - Category 1 (<$150,000)
• 财政年份: 2007
• 资助国家: 加拿大
• 起止时间: 2007-01-01 至 2008-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=366370
• 关键词: Ultralow; freezer; gene; expression; analysis

Understanding the cellular basis of cognition critically requires the assessment of patterns of neural activity over widespread regions of the brain during multiple epochs of behaviour in the same animal at cellular resolution. In fact, a sensitive technique, termed cellular compartment analysis of temporal activity by fluorescence in situ hybridization (catFISH) permits such discrimination by visualizing the transcription of immediate-early genes (IEGs). Neurons engaged in information processing immediately transcribe IEGs that are visible for several minutes within the nucleus. Subsequently, processed mRNA is trans-located to the cytoplasm, peaking in concentration at ~30 min. Because mRNA can be resolved in two independent cellular compartments, it is possible to identify active neurons during two behavioural epochs in the same animal. Moreover, the proportion of cells transcribing IEGs during behaviour equals estimates generated by electrophysiological recording. Thus, like fMRI or PET, catFISH provides large-scale repeated neuronal activity measurements in the same subject, but with the cellular resolution to examine signalling and plasticity in neural networks mediating cognition. This new experimental system allows us to examine signalling and plasticity in neural networks that underlie information processing and storage.  In fact, using this method it has been possible to visualize neurons associated with spatial processing in the hippocampus, movement processing in posterior parietal cortex, and feeding behaviour in the amygdala and orbitofrontal cortex.  Further work in this area, however, critically requires an ultra-low freezer.  The practical constraints inherent in  minimizing experimentally-induced variation in hybridization yield make the storage of tissue for days or weeks unavoidable. During these periods, the availability of storage at temperatures that maintain the integrity of RNA is critical to the quality of the data that is generated.

要理解认知的细胞基础，关键是需要在细胞分辨率下评估同一动物多个行为时期大脑广泛区域的神经活动模式。事实上，一个敏感的技术，称为细胞室分析的时间活动的荧光原位杂交（catFISH）允许这样的歧视，通过可视化的转录立即早期基因（IEG）。参与信息处理的神经元立即转录在细胞核内几分钟内可见的IEGs。随后，处理的mRNA被转移到细胞质中，在约30分钟时浓度达到峰值。由于mRNA可以在两个独立的细胞隔室中分辨，因此可以在同一动物的两个行为时期识别活跃的神经元。此外，在行为过程中转录IEG的细胞比例等于电生理记录产生的估计值。因此，与fMRI或PET一样，catFISH在同一受试者中提供了大规模重复的神经元活动测量，但具有细胞分辨率，以检查介导认知的神经网络中的信号传导和可塑性。这个新的实验系统使我们能够检查神经网络中的信号和可塑性，这些神经网络是信息处理和存储的基础。事实上，使用这种方法，已经可以可视化与海马体中的空间处理、后顶叶皮层中的运动处理以及杏仁核和眶额皮层中的进食行为相关的神经元。然而，在这一领域的进一步工作，最小化实验诱导的杂交产率变化所固有的实际限制使得组织的储存数天或数周不可避免。在这些期间，在保持RNA完整性的温度下储存的可用性对生成的数据的质量至关重要。