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Total RNA Isolation after Laser-capture Microdissection of Human Cervical Squamous Epithelial Cells from Fresh Frozen Tissue

从鲜冻组织中激光捕获显微解剖人宫颈上皮细胞后分离总RNA

SW Saskia M Wilting
RS Renske DM Steenbergen

发布: 2013年08月05日第3卷第15期 DOI: 10.21769/BioProtoc.848 浏览次数: 11457

评审: Lin Fang

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Cover of Oncogene, featuring study using the protocol.

Jan 2013

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Abstract

As most tissue specimens contain a mixture of different cell types including epithelial cells, stromal cells and immune cells, selection of the cells of interest is of utmost importance for the accurate determination of gene/microRNA expression. Laser capture microdissection enables the researcher to obtain homogeneous ultrapure cell selections from heterogeneous starting material. The following protocol was optimized for the isolation of total RNA from cervical (premalignant) squamous epithelial cells from fresh frozen biopsy specimens.

Keywords: LCM (LCM) search RNA (RNA) search Fresh frozen (新鲜冷冻) search

Materials and Reagents

  1. Fresh frozen tissue specimens stored in liquid nitrogen
  2. Tissue-Tek O.C.T. (Sakura Finetek Europe B.V., catalog number: 4583 )
  3. Mayers’ Haematoxylin
  4. Ethanol
  5. Xylene
  6. Liquid nitrogen or dry ice
  7. TRIzol (Life Technologies, catalog number: 15596-026 ) or a preferred alternative RNA isolation method
  8. Glycogen (F. Hoffmann-La Roche, catalog number: 10901393001 )
  9. Chloroform
  10. Isopropanol (Isopropyl alcohol)
  11. RNase-free water

Equipment

  1. 21-26 gauge needle
  2. PEN foil 2.0 UM covered slides (Leica, catalog number: 11505158 )
  3. 50 ml tubes with screw cap (Greiner Bio-one, catalog number:  210261 )
  4. Cryotome (rotary microtome in a frozen section environment)
  5. Leica Laser Microdissection system (AS LMD, model: LMD6500 or LMD7000 )
  6. Centrifuge
  7. -80 °C freezer for sample storage

Procedure

English
中文翻译

文章信息

版权信息

© 2013 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Wilting, S. M. and Steenbergen, R. D. (2013). Total RNA Isolation after Laser-capture Microdissection of Human Cervical Squamous Epithelial Cells from Fresh Frozen Tissue . Bio-protocol 3(15): e848. DOI: 10.21769/BioProtoc.848.

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分类

分子生物学 > RNA > RNA 提取

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