A fluid cover medium provides superior morphology and preserves RNA integrity in tissue sections for laser microdissection and pressure catapulting
A fluid cover medium provides superior morphology and preserves RNA integrity in tissue sections for laser microdissection and pressure catapulting
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DOI：
10.1002/path.1496
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发表时间：
2004-01-01
影响因子：
7.3
通讯作者：
Busch, C
中科院分区：
文献类型：
--
作者：
Micke, P;Bjornsen, T;Busch, C
激光显微切割和加压弹射已成为从几乎所有生物医学研究领域的组织样本中获得均一细胞群的有力工具。分离的细胞随后可用于蛋白质、DNA或RNA的分析。然而，该方法需要物理接触到组织表面，因此切片需要风干并不覆盖。其结果是形态不佳，这严重降低了该技术的潜力，特别是在非均质组织或免疫细胞渗入的组织中。为了克服这一局限性，开发了一种液体覆盖介质，并评估了其对冰冻组织和石蜡包埋组织形态的影响。覆盖介质改善了形态，使其几乎可以与覆盖有玻璃盖片的切片相媲美。此外，激光显微切割过程得到了便利，因为介质允许更大面积的组织被激光压力弹射。无论是蛋白质的分离还是基因组DNA的提取都不会因使用液体覆盖介质而受到不利影响。用TaqMan实时荧光定量聚合酶链式反应和微芯片电泳法检测，覆盖和未覆盖的组织切片之间的RNA数量和完整性没有显著差异。总之，这种方法为激光显微切割和压力弹射技术提供了相当大的改善的形态，而不影响依赖RNA的下游应用。这不仅方便了现有的程序，而且还将扩大应用到需要更好的形态分辨率的组织。版权所有(C)2003 John Wiley Sons，Ltd.
Laser microdissection and pressure catapulting has become a powerful tool to obtain homogeneous cell populations from tissue samples in nearly all fields of biomedical research. The isolated cells can be subsequently used for the analysis of proteins, DNA or RNA. However, the method requires physical access to the tissue surface and the sections therefore need to be air-dried and uncovered. The consequence is poor morphology, which severely reduces the potential of the technique, especially in non-homogeneous tissues or tissues with infiltrating immune cells. To overcome this limitation, a fluid cover medium was developed and the effects on frozen and paraffin wax-embedded tissue morphology were evaluated. The cover medium improved the morphology such that it was almost comparable to sections overlaid with glass coverslips. Moreover, the laser microdissection procedure was facilitated, since the medium allowed larger areas of tissues to be laser pressure-catapulted. Neither the isolation of proteins nor the extraction of genomic DNA was adversely affected by the use of the fluid cover medium. No significant differences in RNA quantity and integrity were detected by TaqMan real-time PCR for GAPDH, and microchip electrophoresis, between covered and uncovered tissue sections. In conclusion, this method provides considerably improved morphology for laser microdissection and pressure catapulting techniques without affecting RNA-dependent downstream applications. This not only facilitates established procedures, but will also extend the application to tissues that require superior morphological resolution. Copyright (C) 2003 John Wiley Sons, Ltd.