激光细胞切割与冰冻切片后高质量 RNA 的分离。

Laser Microdissection of Cells and Isolation of High-Quality RNA After Cryosectioning.

机构信息

Facultad de Ciencias Ambientales y Bioquímica, Universidad de Castilla-La Mancha, Toledo, Spain.

International Research Organization for Advanced Science and Technology (IROAST), Kumamoto University, Kumamoto, 860-8555, Japan.

出版信息

Methods Mol Biol. 2021;2170:35-43. doi: 10.1007/978-1-0716-0743-5_3.

DOI:10.1007/978-1-0716-0743-5_3

PMID:32797449

Abstract

Laser capture microdissection (LCM) has become a powerful technique that allows analyzing gene expression in specific target cells from complex tissues. Widely used in animal research, still few studies on plants have been carried out. We have applied this technique to the plant-nematode interaction by isolating feeding cells (giant cells; GCs) immersed inside complex swelled root structures (galls) induced by root-knot nematodes. For this purpose, a protocol that combines good morphology preservation with RNA integrity maintenance was developed, and successfully applied to Arabidopsis and tomato galls. Specifically, early developing GCs at 3 and 7 days post-infection (dpi) were analyzed; RNA from LCM GCs was amplified and used successfully for microarray assays.

摘要

激光捕获显微切割 (LCM) 已成为一种强大的技术，可用于分析复杂组织中特定靶细胞的基因表达。该技术在动物研究中得到了广泛应用，但在植物研究中应用较少。我们通过分离受根结线虫诱导的复杂肿胀根结构（虫瘿）内的取食细胞（巨型细胞；GCs），将该技术应用于植物-线虫相互作用。为此，开发了一种将良好形态保存与 RNA 完整性保持相结合的方案，并成功应用于拟南芥和番茄虫瘿。具体来说，分析了感染后 3 和 7 天的早期发育 GC；从 LCM GC 中扩增的 RNA 并成功用于微阵列分析。