Bova G Steven, Eltoum Isam A, Kiernan John A, Siegal Gene P, Frost Andra R, Best Carolyn J M, Gillespie John W, Su Gloria H, Emmert-Buck Michael R
Department of Pathology, Institute of Genetic Medicine, The Johns Hopkins Hospital, PELICAN Laboratory, Carnegie 628, Baltimore, MD 21287, USA.
Mol Biotechnol. 2005 Feb;29(2):119-52. doi: 10.1385/MB:29:2:119.
Isolation of well-preserved pure cell populations is a prerequisite for sound studies of the molecular basis of any tissue-based biological phenomenon. This article reviews current methods for obtaining anatomically specific signals from molecules isolated from tissues, a basic requirement for productive linking of phenotype and genotype. The quality of samples isolated from tissue and used for molecular analysis is often glossed over or omitted from publications, making interpretation and replication of data difficult or impossible. Fortunately, recently developed techniques allow life scientists to better document and control the quality of samples used for a given assay, creating a foundation for improvement in this area. Tissue processing for molecular studies usually involves some or all of the following steps: tissue collection, gross dissection/identification, fixation, processing/embedding, storage/archiving, sectioning, staining, microdissection/annotation, and pure analyte labeling/identification and quantification. We provide a detailed comparison of some current tissue microdissection technologies, and provide detailed example protocols for tissue component handling upstream and downstream from microdissection. We also discuss some of the physical and chemical issues related to optimal tissue processing, and include methods specific to cytology specimens. We encourage each laboratory to use these as a starting point for optimization of their overall process of moving from collected tissue to high quality, appropriately anatomically tagged scientific results. In optimized protocols is a source of inefficiency in current life science research. Improvement in this area will significantly increase life science quality and productivity. The article is divided into introduction, materials, protocols, and notes sections. Because many protocols are covered in each of these sections, information relating to a single protocol is not contiguous. To get the greatest benefit from this article, readers are advised to read through the entire article first, identify protocols appropriate to their laboratory for each step in their workflow, and then reread entries in each section pertaining to each of these single protocols.
分离保存完好的纯细胞群体是对任何基于组织的生物学现象的分子基础进行可靠研究的前提条件。本文综述了从组织中分离出的分子获取解剖学特异性信号的当前方法，这是有效关联表型和基因型的基本要求。从组织中分离并用于分子分析的样本质量在出版物中常常被忽视或省略，这使得数据的解释和复制变得困难甚至不可能。幸运的是，最近开发的技术使生命科学家能够更好地记录和控制用于特定分析的样本质量，为该领域的改进奠定了基础。分子研究的组织处理通常涉及以下一些或全部步骤：组织收集、大体解剖/识别、固定、处理/包埋、储存/存档、切片、染色、显微切割/注释以及纯分析物标记/识别和定量。我们对一些当前的组织显微切割技术进行了详细比较，并提供了显微切割上下游组织成分处理的详细示例方案。我们还讨论了与最佳组织处理相关的一些物理和化学问题，并包括了细胞学标本特有的方法。我们鼓励每个实验室将这些作为优化其从收集组织到获得高质量、具有适当解剖学标记的科学结果的整体过程的起点。在当前生命科学研究中，优化方案存在效率低下的问题。该领域的改进将显著提高生命科学的质量和生产力。本文分为引言、材料、方案和注释部分。由于每个部分都涵盖了许多方案，与单个方案相关的信息并不连贯。为了从本文中获得最大收益，建议读者先通读全文，为其工作流程中的每个步骤确定适合其实验室的方案，然后重新阅读每个部分中与这些单个方案相关的条目。
