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Laser Microdissection‐Mediated Isolation and In Vitro Transcriptional Amplification of Plant RNA

Michael J. Scanlon1,  Kazuhiro Ohtsu2,  Marja C.P. Timmermans3,  Patrick S. Schnable2

1Cornell University, Ithaca, New York
2Iowa State University, Ames, Iowa
3Cold Spring Harbor Laboratory, Cold Spring Harbor, New York



Publication Name: 
Current Protocols in Molecular Biology
Unit Number: 
Unit 25A.3
DOI: 
10.1002/0471142727.mb25a03s87
Online Posting Date: 
July, 2009
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Abstract

Protocols for laser microdissection and linear amplification of RNA from fixed, sectioned plant tissues are described. When combined with quantitative RT-PCR, microarray analysis, or RNA-sequencing, these procedures enable quantitative analyses of transcript accumulation from microscopic quantities of specific plant organs, tissues, or single cells. Curr. Protoc. Mol. Biol. 87:25A.3.1-25A.3.15 © 2009 by John Wiley & Sons, Inc.

Keywords: laser microdissection; plants; RNA amplification; transcriptomics

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Laser Microdissection of Plant RNA
  • Basic Protocol 2: In Vitro Transcriptional Amplification of RNA
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Laser Microdissection of Plant RNA

 Materials
  • Maize seedlings 14 days post-germination
  • Acetone (100%, Fisher Scientific), ice-cold and room temperature
  • Ice
  • Xylene (Fisher Scientific)
  • Diethylpyrocarbonate (DEPC; Sigma)
  • 100% ethanol
  • Mineral oil (optional)
  • PicoPure RNA Isolation Kit (Arcturus)
  • Razor blade (single-edged)
  • Petri dishes (glass)
  • Scintillation vials (20 ml, Fisher Scientific)
  • Vacuum apparatus
  • Rotator (e.g., Ted Pella)
  • Paraplast chips (Paraplast +, 56°C, Oxford Labware)
  • Oven preset to 60°C
  • Gradient metal warming plate (a paraffin-embedding center can be used if one is available)
  • Metal weighing dish
  • Tweezers or paintbrush (fine point)
  • Paraffin embedding rings (Simport)
  • Paraffin clear base molds (Surgipath)
  • Plastic bags
  • Rotary microtome
  • Probe-on-Plus slides (Fisher Scientific) or PEN Membrane Slides (P.A.L.M. microbeam)
  • Slide-warming tray (Fisher Scientific)
  • Paper towels
  • Dissecting microscope
  • PALM MicroBeam System (Carl Zeiss)
  • PALM adhesive cap tubes (Carl Zeiss) or 0.5-ml centrifuge tubes with caps

NOTE: Work in a fume hood until samples are securely capped and placed at 4°C. Keep fixative cold at all times to ensure slow penetration of fixative.

Basic Protocol 2: In Vitro Transcriptional Amplification of RNA

 Materials
  • T7-oligo(dT) primer (0.5 µg/µl):
    • (5¢TCTAGTCGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGTTTTTTTTTTTTTTTTTTTTT-3¢)
  • RNA extracted from laser microdissection (LM) sample (see Basic Protocol 1)
  • Diethylpyrocarbonate (DEPC; Sigma)
  • dNTP mix (10 mM, Intermountain Scientific)
  • SuperScript II Reverse Transcriptase (200 U/µl, Invitrogen) containing:
    • 5× first-strand buffer
    • 0.1 M DTT
  • RNaseOUT Recombinant Ribonuclease Inhibitor (40 U/µl, Invitrogen)
  • T4 gene 32 protein (5 µg/µl, USB)
  • E. coli DNA polymerase I (10 U/µl, New England Biolabs) containing:
    • 10× DNA polymerase I buffer
  • -Nicotinamide adenine dinucleotide hydrate (-NAD+; 260 µM, min. 98% from yeast, Sigma)
  • Ribonuclease H (RNase H; 2 U/µl, Invitrogen)
  • E. coli DNA ligase (10 U/µl, New England Biolabs)
  • T4 DNA polymerase (3 U/µl, New England Biolabs)
  • Phenol (Saturated, Fisher Scientific):
    • pH 6.6, BP1750I-400 (for step 10)
    • pH 4.3, BP1751I-400 (for step 18)
  • Chloroform (~0.75% ethanol as preservative, Technical grade, Fisher Scientific)
  • QIAquick PCR Purification Kit including:
    • Qiagen 250 columns
    • Buffer PB
    • Buffer PE
    • Bufffer EB
  • Sodium acetate (100 mM, pH 5.2, certified ACS, Fisher Scientific)
  • MEGAscript T7 Kit (Ambion) including:
    • rNTP solutions
    • 10× reaction buffer
    • T7 RNA polymerase enzyme mix
    • RNase-free DNase I
  • Nuclease-free H2O
  • RNeasy Mini Kit (50 columns; Qiagen) includes:
    • 1.5- and 2.0-ml collection tubes
    • RNase-free reagents and buffers (including Buffer RLT and Buffer RPE)
  • Ethanol (Absolute, Aaper Alcohol)
  • Random hexamer primer (1 µg/µl, Roche Diagnostics)
  • Microcentrifuge tubes (nuclease-free)
  • Heating block or water bath preset to 16°C, 37°C, 42°C, 65°C, 70°C, 95°C
  • Concentrator/evaporator
  • Vortex
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Figures

  • Figure 25A.3.1
    Flowchart of the use of laser microdissection for analysis of transcript accumulation within plant tissue microdomains. In this example, mesophyll cells are microdissected (green arrows) from transverse sections (10-µm) of mature rosette leaves of Arabidopsis thaliana. Images of Arabidopsis leaf sections were provided by K. Petsch, Cornell University; agarose gel image of IVT-amplified RNA is kindly provided by X. Zhang, University of Georgia.

    View Image

Literature Cited

Literature Cited
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    Klink, V.P., Alkharouf, N., MacDonald, M., and Matthews, B. 2005. Laser capture microdissection (LCM) and expression analyses of Glycine max (soybean) syncytium containing root regions formed by the plant pathogen Heterodera glycines (soybean cyst nematode). Plant Mol. Biol. 59:965-979.
    Luo, L., Salunga, R.C., Guo, H., Bittner, A., Joy, K.C., Galindo, J.E., Xiao, H., Rogers, K.E., Wan, J.S., Jackson, M.R., and Erlander, M.G. 1999. Gene expression profiles of laser-captured adjacent neuronal subtypes. Nat. Med. 5:117-122.
    Luzzi, V., Mahadevappa, M., Raja, R., Warrington, J.A., and Watson, M.A. 2003. Accurate and reproducible gene expression profiles from laser capture microdissection, transcript amplification, and high-density oligonucleotide microarray analysis. J. Mol. Diagn. 5:9-14.
    Mustafa, D., Kros, J.M., and Luider, T. 2008. Combining laser capture microdissection and proteomics techniques. Methods Mol. Biol. 428:159-178.
    Nakazono, M., Qiu, F., Borsuk, L.A., and Schnable, P.S. 2003. Laser-capture microdissection, a tool for the global analysis of gene expression in specific plant cell types: Identification of genes expressed differentially in epidermal cells or vascular tissues of maize. Plant Cell. 15:583-596.
    Nelson, T., Tausta, S.L., Gandotra, N., and Liu, T. 2006. Laser microdissection of plant tissue: What you see is what you get. Annu. Rev. Plant Biol. 57:181-201.
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    Schneider, J., Buness, A., Huber, W., Volz, J., Kioschis, P., Hafner, M., Poustka, A., and Sültmann, H. 2004. Systematic analysis of T7 RNA polymerase–based in vitro linear RNA amplification for use in microarray experiments. BMC Genomics 5:29.
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    Wu, Y., Llewellyn, D.J., White, R., Ruggiero, K., Al-Ghazi, Y., and Dennis, E.S. 2007. Laser capture microdissection and cDNA microarrays used to generate gene expression profiles of the rapidly expanding fibre initial cells on the surface of cotton ovules. Planta. 22:1475-1490.
    Yu, Y., Lashbrook, C.C., and Hannapel, D.J. 2007. Tissue integrity and RNA quality of laser microdissected phloem of potato. Planta 226:797-803.
    Zhang, X., Madi, S., Borsuk, L., Nettleton, D., Elshire, R.J., Buckner, B., Janick-Buckner, D., Beck, J., Timmermans, M., Schnable, P.S., and Scanlon, M.J. 2007. Laser microdissection of narrow sheath mutant maize uncovers novel gene expression in the shoot apical meristem. PLoS Genet. 3:e101.
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 Internet Resources
    http://www.palm-microlaser.com/dasat/index.php cid=100113&conid=0&sid=dasat

Offers product information for PALM MicroLaser Systems at Carl Zeiss.

     
 
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