The CLIP‐CHIP Oligonucleotide Microarray: Dedicated Array for Analysis of All Protease, Nonproteolytic Homolog, and Inhibitor Gene Transcripts in Human and Mouse

Reinhild Kappelhoff1, Chris Overall1

1 University of British Columbia, Vancouver, British Columbia, Canada
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 21.19
DOI:  10.1002/0471140864.ps2119s56
Online Posting Date:  April, 2009
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Abstract

The CLIP‐CHIP oligonucleotide microarray allows the analysis of mRNA transcript levels in a tissue sample for all proteases, nonproteolytic homologs, and protease inhibitors of the human and mouse genome. In the protocol presented in this unit, total RNA is extracted from a tissue, and the resulting mRNA is reverse transcribed into cDNA and dsDNA and then amplified in an in vitro transcription reaction. The amplified antisense RNA is labeled with a fluorescent dye and hybridized to the CLIP‐CHIP, which contains unique oligonucleotides that are specifically designed for the protease, nonproteolytic homologs, protease inhibitors, and control samples. After hybridization, the fluorescence intensity of each spot is measured, thus identifying mRNA transcripts that are expressed and allowing basic quantification of expressed transcripts. Curr. Protoc. Protein Sci. 56:21.19.1‐21.19.16. © 2009 by John Wiley & Sons, Inc.

Keywords: mouse and human oligonucleotide microarray; linear amplification; aRNA; direct aRNA labeling

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • Cells of interest growing in tissue culture or whole tissue of interest, and appropriate control cells or tissue
  • Phosphate‐buffered saline (PBS; appendix 2E)
  • RNeasy Mini Kit (Qiagen), including:
    • RNeasy Mini spin columns
    • RLT buffer containing 1% 2‐mercaptoethanol
    • RW1 buffer
    • RPE buffer
    • Nuclease‐free water
  • Total RNA isolation reagent: TRIzol (Invitrogen) or Tri Reagent (Sigma)
  • 70% (v/v) ethanol (made with RNase‐free water)
  • RNase‐free DNase set (Qiagen), including:
    • RDD buffer
    • DNase I (30 Kunitz U/µl)
  • 1.2% agarose gel in 1× TAE buffer ( appendix 2E; made under RNase‐free conditions)
  • Agilent Bioanalyzer 2100 and a RNA6000 LabChip Kit (optional)
  • MessageAmp II aRNA amplification kit (Ambion), including:
    • 100 ng/µl T7‐oligo (dT) 24‐promotor primer)
    • 10× first‐strand buffer
    • dNTP mix (2.5 mM each dNTP)
    • 10 U/µl RNase inhibitor
    • 200 U/µl ArrayScript
    • 10× second‐strand buffer
    • 5 U/µl DNA polymerase
    • 10 U/µl RNase H
    • cDNA filter cartridges
    • cDNA binding buffer
    • cDNA wash buffer
    • 10× T7‐Reaction Buffer
    • 75 mM T7 ATP
    • 75 mM T7 CTP
    • 75 mM T7 GTP
    • 75 mM T7 UTP
    • T7 Enzyme Mix
    • aRNA filter cartridges
    • aRNA binding buffer
    • aRNA wash buffer
    • 5 M ammonium acetate
    • Nuclease‐free water
  • 100% ethanol (ACS grade)
  • 70% (v/v) ethanol made with nuclease‐free H 2O (see recipe for DEPC‐treated H 2O), −20°C
  • ULS aRNA fluorescent labeling kit (Kreatech Biotechnology, http://www.kreatech.com), including:
    • Cy5‐ULS
    • Cy3‐ULS
    • 10× labeling Buffer
    • KREApure spin columns and 2‐ml collection tubes
    • KREAblock
  • Prehybridization buffer (see recipe)
  • Isopropanol
  • RNA fragmentation reagents (Ambion), including:
    • 10× fragmentation buffer (buffered zinc solution)
    • 200 mM EDTA, pH 8
  • 2× hybridization buffer (see recipe), preheated to 42°C
  • 3× SSC ( appendix 2E; prepare under RNase‐free conditions)
  • Wash buffer 1: 1× SSC ( appendix 2E) containing 0.2% SDS (add from 10% w/v SDS stock), filtered through 0.2‐µm filter and preheated to 42°C
  • Wash buffer 2: 0.1× SSC ( appendix 2E) containing 0.2% SDS (add from 10% w/v SDS stock), filtered through 0.2‐µm filter and preheated to 42°C
  • Wash buffer 3: 0.1× SSC ( appendix 2E), preheated to 42°C
  • 6‐well tissue culture plate
  • Nonstick RNase‐free microcentrifuge tubes
  • QIAshredder spin columns (Qiagen) or 20‐G needles and syringes
  • Rotor‐stator tissue homogenizer with probe either autoclaved or treated with anti‐RNase solution
  • Spectrophotometer (e.g., NanoDrop ND‐1000A UV‐vis)
  • Hybridization oven, thermal cycler, or temperature‐controlled heat blocks capable of chilling to 16°C (cover block with tin foil) and heating to 42°, 48°, 55°, 60°, 70°, 85°, and 95°C
  • Aminosilane microarray slides with oligonucleotides of interest (CLIP‐CHIP, Overall Lab at The University of British Columbia; http://www.clip.ubc.ca)
  • Coplin jars
  • 50‐ml conical polypropylene centrifuge tubes
  • LifterSlips (coverslip with Teflon spacer giving 0.75‐mm gap; Erie Scientific): 22 × 25–mm to cover individual subarrays or 22 × 60–mm to cover whole array
  • Microarray hybridization chamber (Corning or equivalent) or empty pipet tip box
  • Zip‐Lock bags
  • Black glass slide storage boxes
  • Microarray scanner (ScanArray from Perkin‐Elmer; arrayWoRx from Applied Precision; GenePix from Axon Instruments; or 428 Scanner from MWG)
  • Microarray analysis software (see )
  • Additional reagents and equipment for spectrophotometric determination of nucleic acid concentration ( appendix 4K) and agarose gel electrophoresis ( appendix 4F)
NOTE: All work should be done in an RNase‐free environment. Clean the surface of the workspace, pipets, and pipet‐tip boxes with an anti‐RNase chemical—e.g., 3% H 2O 2, RNaseZAP (Ambion), or RNaseAway (Molecular Bioproducts). Use disposable gloves and new, one‐time‐use disposable plasticware. RNase/DNase‐free tubes should be used and solutions should be prepared with RNase‐free water (e.g., VWR or Fisher, or see recipe for DEPC‐treated water). Pipet tips with filters should be employed to prevent RNase contamination. Use nonstick, RNase‐free microcentrifuge tubes when working with small amounts of RNA solution or any other liquids, to maximize recovery. Prehybridization and wash buffers used for the microarray slides do not have to be prepared with nuclease‐free water.
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Figures

Videos

Literature Cited

   Brown, T., Mackey, K., and Du, T. 2004. Analysis of RNA by northern and slot blotting. Curr. Protoc. Mol. Biol. 67:4.9.1‐4.9.19.
Key References
   Overall, C.M., Tam, E.M., Kappelhoff, R., Connor, A., Ewart, T., Morrison, C.J., Puente, X.S., Lopez‐Otin, C., and Seth, A. 2004. Protease degradomics: Mass spectrometry discovery of protease substrates and the CLIP‐CHIP, a dedicated DNA microarray of all human proteases and inhibitors. Biol. Chem. 385:493‐504.
  The first paper in which the CLIP‐CHIP was mentioned. The method described in this paper was completely changed into the method described in this unit. The paper presents CLIP‐CHIP as a new customized microarray dedicated to all human proteases and inhibitors. Since then, the authors have also printed and designed the mouse CLIP‐CHIP.
Internet Resources
  http://www.clip.ubc.ca.
  Provides a current list of proteases and inhibitors present on the human or mouse or human and mouse CLIP‐CHIP This site also includes gene identity files needed to analyze CLIP‐CHIP data and presents updates for the microarray procedures.
  http://www.uniovi.es/degradome
  This Web page from Dr. Carlos López‐Otín and Dr. Xose Puente compares all proteases and inhibitors in human, mouse, and rat. When possible, every protease and inhibitor is linked to its MEROPS (http://merops.sanger.ac.uk) database entry.
  http://www.tm4.org
  TM4 microarray software suite from the Institute of Genomic Research.
  http://www.pathogenomics.ca
  ArrayPipe microarray software from the Institute for Pathogenomics of Innate Immunity.
  https://carmaweb.genome.tugraz.at/carma/
  CARMAweb microarray software from Tyrolean Cancer Research Institute
  http://rana.lbl.gov
  Scanalyze microarray software from the Eisen Lab.
  http://www.biodiscovery.com
  Imagene: commercial microarray software from BioDiscovery.
  http://www.chem.agilent.com
  GeneSpring: commercial microarray software from Agilent.
  http://www.moleculardevices.com
  GenePix: commercial microarray software from Molecular Devices.
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