Multi‐Color FISH Techniques

Jane Bayani1, Jeremy Squire1

1 Ontario Cancer Institute, Toronto
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 22.5
DOI:  10.1002/0471143030.cb2205s24
Online Posting Date:  October, 2004
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Abstract

Traditional FISH analysis has employed, at most, two colors of detection, a red‐fluorescing fluorochrome and a green‐fluorescing fluorochrome. The improvements in fluorescent imaging and development of heartier fluorochromes/dyes have enabled investigators to use several different DNA probes in one experiment. This may involve all or combinations of locus‐specific probes and chromosome paints. The value of such experiments lies in the investigator obtaining far more information from one specific cell at one time, rather then carrying out separate experiments on multiple specimens prepared from the same sample, then extrapolating results. The generic term for multi‐color FISH assays is M‐FISH, however, the technologies behind the manner in which the fluorochrome information is generated has spawned two different M‐FISH systems: spectral karyotyping (SKY) and M‐FISH. For both assays, the experimental procedures are identical: commercially available probes for all 24 (human) chromosomes are differentially labeled according to a labeling scheme and hybridized to metaphase spreads for 24 to 48 hr, followed by post‐hybridization washes and, if required, antibody detection. The difference lies in the imaging: spectral karyotyping identifies the differentiation of the chromosomes based on their spectral properties, whereas M‐FISH identifies the differentiation of the chromosomes based on that fluorochrome's presence or absence when visualized with specific filters. The resulting analysis for both methods is the same, revealing hidden translocations and insertions as well as the chromosomal components of marker chromosomes.

Keywords: FISH; SKY; M‐FISH; hybridization; whole‐chromosome paints; locus‐specific probe

     
 
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Table of Contents

  • Basic Protocol 1: Labeling Whole‐Chromosome DNA Probes for Multi‐Color FISH Assays
  • Basic Protocol 2: In Situ Hybridization for Spectral Karyotyping (SKY)
  • Alternate Protocol 1: In Situ Hybridization for M‐FISH Karyotyping
  • Alternate Protocol 2: Pretreatment of Previously G‐Banded Slides for SKY OR M‐FISH
  • Alternate Protocol 3: FISH Analysis Using Locus‐Specific or Chromosome Painting Probes Following SKY/M‐FISH Hybridization
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Labeling Whole‐Chromosome DNA Probes for Multi‐Color FISH Assays

  Materials
  • Template DNA (flow‐sorted chromosomes in a volume of 32.0 µl; Gray et al., )
  • 5× buffer D (accompanying 10 mM dNTP from Invitrogen)
  • 10 mM dNTP nucleotide mix (see recipe)
  • 5 µM 5′‐CCGACTCGAGNNNNNNATGTGG‐3′ primer
  • 15 U/µl Super Taq polymerase (CPG)
  • 5× loading buffer (see recipe)
  • 2% (w/v) agarose gel with ethidium bromide (see recipe)
  • 5 U/µl AmpliTaq polymerase (Perkin Elmer)
  • 10× PCR buffer
  • 25 mM MgCl 2
  • 2 mM dNTP for secondary amplification (see recipe)
  • 2 mM dNTP for labeling (see recipe)
  • 100 µM Rhodamine 110 (Perkin Elmer)
  • 1 mM FITC
  • 1 mM Texas Red (see recipe for x‐dUTPs)
  • 50 mM biotin (Boehringer Mannheim)
  • 50 mM digoxygenin (Boehringer Mannheim)
  • 1 mM Spectrum green (see recipe for x‐dUTPs)
  • 1 mM Spectrum orange (see recipe for x‐dUTPs)
  • 1 mM Spectrum aqua (see recipe for x‐dUTPs)
  • 1 mM Spectrum gold (see recipe for x‐dUTPs)
  • 1 mM Spectrum far red (see recipe for x‐dUTPs)
  • 1 mg/ml human Cot‐1 DNA (GIBCO)
  • 10.0 mg/ml sonnicated salmon sperm DNA (Invitrogen)
  • 3 M sodium acetate ( appendix 2A)
  • 100% and 70% ethanol
  • Hybridization buffer (see recipe)
  • PCR tubes
  • PCR thermal cycler
  • Gel system and power source
  • 0.5‐ and 2‐ml microcentrifuge tubes
  • Refrigerated centrifuge
NOTE: Use aseptic technique throughout. Be sure to change pipet tips for each reagent and before addition to each tube.

Basic Protocol 2: In Situ Hybridization for Spectral Karyotyping (SKY)

  Materials
  • Metaphase slides (unit 22.2)
  • 0.01 M HCl (see recipe), prewarmed to 37°C
  • 10% (w/v) pepsin (see recipe)
  • 1× PBS
  • 1% formaldehyde/1× PBS/50 mM MgCl 2 (see recipe)
  • 70% formamide/2× SSC (see recipe), prewarmed to 72°C
  • 70%, 80%, and 100% ethanol
  • SKY kit (Applied Spectral Imaging) containing probe cocktail, block reagents, detection reagents, and counterstain or in‐house custom‐made probes in hybridization solution (see protocol 1)
  • Rubber cement
  • 50% formamide/2× SSC, 45°C (3 Coplin jars)
  • 1× SSC (see recipe), 45°C (3 Coplin jars)
  • 0.1%Tween‐20/4× SSC (see recipe), 45°C (7 Coplin jars)
  • Blocking solution (see recipe)
  • Avidin‐Cy5 antibodies (see recipe for antibodies)
  • Mouse anti‐digoxigenin (see recipe for antibodies)
  • Cy5.5 anti‐mouse antibodies (see recipe for antibodies)
  • DAPI/antifade counterstain (see recipe)
  • Clear nail polish (do not use if planning to re‐probe slide)
  • Phase‐contrast microscope
  • 37°C oven
  • Fume hood
  • 22 × 22–mm coverslips
  • Hybridization container
  • Fluorescent microscope and associated Applied Spectral Imaging Acquisition hardware and software (Applied Spectral Imaging)

Alternate Protocol 1: In Situ Hybridization for M‐FISH Karyotyping

  Materials
  • Chromosome spreads (unit 22.2)
  • 70%, 80%, and 100% ethanol
  • RNase A working solution (see recipe)
  • 2× SSC (see recipe)
  • 10% pepsin stock (see recipe)
  • 0.01 M HCl (see recipe), 37°C
  • 1× PBS (2 coplin jars)
  • 1% formaldehyde/1× PBS/50 mM MgCl 2 (see recipe)
  • 70% formamide/2× SSC (see recipe), 72°C
  • Commercially available M‐FISH probes (Vysis) or in‐house custom probes
  • 0.4× SSC/0.3% NP‐40 (see recipe), 73°C
  • 2× SSC/0.1% NP‐40 (see recipe)
  • DAPI/antifade counterstain (DAPI III, Vysis or see recipe)
  • 50% formamide/2× SSC (see recipe), 45°C
  • 1× SSC (see recipe), 45°C
  • 0.1% Tween‐20/4× SSC (see recipe), 45°C
  • Clear nail polish (do not use if planning to re‐probe slide)
  • Phase‐contrast microscope
  • Glass coverslips
  • 37°C dry oven or incubator
  • Fume hood

Alternate Protocol 2: Pretreatment of Previously G‐Banded Slides for SKY OR M‐FISH

  Materials
  • G‐banded metaphase slides (unit 22.3)
  • Xylene
  • 100% methanol
  • 70%, 80%, and 100% ethanol
  • 1× PBS
  • 1× PBS/50 mM MgCl 2
  • 1%formaldehyde/1× PBS/50 mM MgCl 2 (see recipe)
  • 70% formamide/2× SSC (see recipe), 72°C
  • Fume hood

Alternate Protocol 3: FISH Analysis Using Locus‐Specific or Chromosome Painting Probes Following SKY/M‐FISH Hybridization

  Materials
  • Hybridized SKY or M‐FISH slides (see protocol 2 or protocol 3, respectively)
  • 2× SSC (see recipe)
  • 0.1% Tween‐20/4× SSC (see recipe)
  • 70%, 90%, and 100% ethanol
  • 70% formamide/2× SSC (see recipe), 75°C
  • Labeled DNA probe (as prepared in unit 22.4 or from a commercial supplier)
  • Rubber cement
  • Coverslips
  • Hybridization box
  • 37°C dry oven or incubator
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Figures

  •   FigureFigure 22.5.1 Schematic representation of spectral karyotyping image acquisition and image analysis. The metaphase, following hybridization with the SKYPaints is visualized by fluorescence microscopy. The light passes into the optical head containing the interferometer where 100 different images are taken. This information is Fourier transformed and processed using the SKYView software for analysis. (Adapted from the ASI manual.)
  •   FigureFigure 22.5.2 SKY analysis of a primary ovarian carcinoma. (A) Typical representations of SKY analysis. An inverted DAPI image reveals banding patterns similar to G‐banding. (B) Red‐Green‐Blue (RGB) image of the hybridized spread as it would appear with the probe colors. (C) Classified or pseudo‐colored image to help identify chromosomal aberrations not readily evident in the RGB image. Each chromosome is identified by a specific pseudo‐color. A change in color along the length of a chromosome indicates a different chromosome. (D) SKY karyotype showing each representation of the chromosomes: RGB, inverted DAPI, and classified images.
  •   FigureFigure 22.5.3 Schematic representation of M‐FISH image acquisition and image analysis. M‐FISH experiments involve the imaging of each dye or fluorochrome using a specific filter. The images are then stacked to produce the final merged image. (Adapted from the Vysis manual.)
  •   FigureFigure 22.5.4 MBand using the 11Cyte probe and imaging system from Metasystems. (A) Metaphase spread from a patient possessing an inversion on chromosome 11, hybridized with the 11Cyte chromosome paint. Five different dyes are used to create the painting probe along the length of the chromosome in a specific order. (B) Ideogram of chromosome 11 showing the color order of the dyes along the length of the chromosome and the band regions they span. (C) The normal and inverted chromosome 11.
  •   FigureFigure 22.5.5 The results of sequential G‐banding (A) and SKY (B) analysis of a rhabdomyoscaroma cell line.
  •   FigureFigure 22.5.6 The results of sequential G‐banding (A), SKY (B), and locus‐specific FISH (C) for the BCR‐ABL translocation in a CML sample.

Videos

Literature Cited

Literature Cited
   Gray, J.W., Lukas, J., Peters, D., Pinkel, D., Trask, B., Van den Engh, G., and Van Dilla, M. 1986. Flow karyotyping and sorting of human chromosomes. In Proceedings of Cold Spring Harbor Symposium in Quantitative Biology 51 Pt. 1:141‐149. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
   Malik, Z., Dishi, M., and Garini, Y. 1996. Fourier transform multipixel spectroscopy and spectral imaging of protoporphyrin in single melanoma cells. Photochem. Photobiol. 63:608‐614.
   Schrock, E., du Manoir, S., Veldman, T., Schoell, B., Wienberg, J., Ferguson‐Smith, M.A., Ning, Y., Ledbetter, D.H., Bar‐Am, I., Soenksen, D., Garini, Y., and Ried, T. 1996. Multicolor spectral karyotyping of human chromosomes. Science 273:494‐497.
   Speicher, M.R., Gwyn Ballard, S., and Ward, D.C. 1996. Karyotyping human chromosomes by combinatorial multi‐fluor FISH. Nat. Genet. 12:368‐375.
   Telenius, H., Pelmear, A.H., Tunnacliffe, A., Carter, N.P., Behmel, A., Ferguson‐Smith, M.A., Nordenskjold, M., Pfragner, R., and Ponder, B.A. 1992. Cytogenetic analysis by chromosome painting using DOP‐PCR amplified flow‐sorted chromosomes. Genes Chromosomes Cancer. 4:257‐263.
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