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

Divya Chandran1, Michael J. Scanlon2, Kazuhiro Ohtsu3, Marja C.P. Timmermans4, Patrick S. Schnable3, Mary C. Wildermuth5

1 Regional Center for Biotechnology, Faridabad, 2 Cornell University, Ithaca, New York, 3 Iowa State University, Ames, Iowa, 4 Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 5 University of California, Berkeley, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 25A.3
DOI:  10.1002/0471142727.mb25a03s112
Online Posting Date:  October, 2015
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Abstract

Laser microdissection of cells allows for isolation of specific cells of interest for downstream analyses including transcriptional profiling. Plant cells present unique challenges for laser microdissection due to their cellulosic cell walls and large vacuoles. Here we present protocols for plant tissue preparation, laser microdissection of select plant cells, and linear amplification of RNA from dissected cells. Linear amplification of RNA from dissected cells allows sufficient RNA for subsequent quantitative analysis by RT‐PCR, microarray, or RNA sequencing. © 2015 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 Cells using Acetone Fixation, Paraffin Embedding, and the PALM LMPC System
  • Basic Protocol 2: Laser Microdissection of Plant Cells using Buffer Without Fixative, Modified Microwave Paraffin Embedding, and Leica LMD System
  • Basic Protocol 3: In Vitro Transcriptional Amplification of RNA
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Laser Microdissection of Plant Cells using Acetone Fixation, Paraffin Embedding, and the PALM LMPC System

  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
  • PicoPure RNA Isolation Kit (Arcturus)
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: Laser Microdissection of Plant Cells using Buffer Without Fixative, Modified Microwave Paraffin Embedding, and Leica LMD System

  Materials
  • RNaseZAP (Ambion)
  • Diethylpyrocarbonate (DEPC; Sigma)
  • Fully expanded mature leaves from 4‐week old Arabidopsis plants
  • Sorensen's phosphate buffer solution: 10 mM sodium phosphate buffer, pH 7.2 ( appendix 22) made using RNase‐free H 2O (see recipe), prepared fresh daily and chilled to 4°C
  • RNase‐free (DEPC‐treated) H 2O (see recipe)
  • 30%, 50%, 70%, 95%, and 100% high‐purity solvent–grade ethanol (prepared with DEPC‐treated water)
  • Safranin‐O (Sigma‐Aldrich) dissolved as 1% (w/v) in 100% ethanol.
  • 1:1 ethanol:isopropanol (high purity solvent–grade)
  • 100% isopropanol (high purity solvent–grade)
  • Paraffin wax (Paraplast X‐TRA, Fisher Scientific)
  • Desiccant
  • Xylene, histological/cytological grade
  • PicoPure RNA Isolation Kit (Arcturus)
  • Ribo Green quantification kit (optional; e.g., Life Technologies)
  • Razor blade (single‐edged)
  • Glass plate (treated with RNaseZAP)
  • 15‐ml clear glass threaded vials
  • Paper for sample label
  • Paraffin wax dispenser (Ted Pella)
  • Gradient metal warming plate (a paraffin‐embedding center can be used if one is available; Leica EG1150H)
  • BP111RS tissue processing microwave oven (Microwave Research and Applications; note that this is not a standard kitchen microwave)
  • Makeshift water bath to be placed in microwave oven (microwavable plastic container that holds plastic tube rack and is filled with tap water)
  • Aluminum weighing dish with handle (Fisher Scientific) for use as embedding mold
  • Long straight needle
  • Polyethylene napthalate (PEN) slides, 2.0 μm (JH Technologies Inc.)
  • 100°C oven
  • Wooden blocks [approximately 2 cm (l) × 1.5 cm (w) × 1 cm (d)]
  • Spatula
  • Rotary microtome
  • Paintbrushes (fine point)
  • Hot plate
  • Oven capable of 42°C and 100°C
  • Coplin jars
  • Glass staining dish with cover
  • Glass slide rack with handle
  • Leica AS LMD System (Leica Microsystems GmbH)
  • 0.2‐ml PCR tubes, RNase‐free, compatible with Leica AS LMD system
  • NanoDrop microspectrophotometer (optional)
NOTE: Specific tubes are required that are compatible with the Leica AS LMD. The suppliers change, so confirm compatibility of tubes and obtain supplier information from Leica prior to ordering.

Basic Protocol 3: 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 Protocols protocol 11 and protocol 22)
  • Diethylpyrocarbonate (DEPC; Sigma)
  • dNTP mix (10 mM, Intermountain Scientific)
  • SuperScript II Reverse Transcriptase (200 U/μl, Invitrogen) containing:
    • 5× first‐strand buffer
    • 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
  • β‐Nicotinamide adenine dinucleotide hydrate (β‐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 H 2O
  • RNeasy Mini Kit (50 columns; Qiagen) including:
    • 1.5‐ and 2.0‐ml collection tubes
    • RNase‐free reagents and buffers (including Buffer RLT and Buffer RPE)
  • Ethanol (Absolute, Pharmaco AAPER)
  • Random hexamer primer (1 μg/μl, Roche Diagnostics)
  • Microcentrifuge tubes (nuclease‐free)
  • Thermocyler
  • SpeedVac concentrator/evaporator
  • Vortex
NOTE: Use RNase‐free or DEPC‐treated water in all recipes and protocol steps.NOTE: All centrifugation steps are performed in a benchtop microcentrifuge at room temperature.
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Figures

Literature Cited

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Internet Resources
  http://www.zeiss.com/microscopy/en_de/products/laser‐microdissection/microbeam.html
  The above Web sites provide current product information for PALM MicroLaser Systems from Carl Zeiss and Laser Microdissection Systems from Leica, respectively.
  http://www.leica‐microsystems.com/products/light‐microscopes/life‐science‐research/laser‐microdissection/details/product/leica‐lmd7000/
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