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

Saskia M Wilting; Renske DM Steenbergen

doi:10.21769/BioProtoc.848

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Peer-reviewed

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 email

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: 10910

评审: Lin Fang

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参见作者原研究论文

The authors used this protocol in:

Cover of Oncogene, featuring study using the protocol.

Jan 2013

实验方案合集

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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)

RNA (RNA)

Fresh frozen (新鲜冷冻)

Materials and Reagents

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

Equipment

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

Procedure

English

中文翻译

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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.