Quantitative Fluorescence In Situ Hybridization (Q‐FISH)

Steven S.S. Poon1, Peter M. Lansdorp1

1 BC Cancer Research Centre and University of British Columbia, Vancouver, Canada
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 18.4
DOI:  10.1002/0471143030.cb1804s12
Online Posting Date:  November, 2001
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Abstract

This unit describes a quantitative technique for measuring the lengths of telomere repeat sequences in individual chromosomes from single metaphase cells. The technique is based on fluorescence in situ hybridization (FISH) adapted for use with peptide nucleic acid (PNA) probes. PNA is an example of novel synthetic oligonucleotide “mimetic” which has a higher affinity than regular oligonucleotide (RNA or DNA) probes for complementary single‐strand (ss) DNA sequences. PNA oligonucleotides have excellent penetration properties due to their small size (typically 15 to 18‐mers) and can be directly labeled with fluorochromes. These properties have been exploited to develop quantitative fluorescence in situ hybridization (Q‐FISH) onto denatured single‐stranded chromosomal DNA target sequences. The latter can be present in preparations of fixed metaphase cells on slides (Q‐FISH) or in heat‐treated (interphase) cells in suspension (flow‐FISH).

     
 
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Table of Contents

  • Basic Protocol 1: Using PNA Probes to Visualize Telomeres in Metaphase Spreads
  • Support Protocol 1: Image Capture
  • Support Protocol 2: Image Analysis
  • Support Protocol 3: System Calibration
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Using PNA Probes to Visualize Telomeres in Metaphase Spreads

  Materials
  • Cells of interest
  • Cell‐specific tissue culture medium supplemented with growth factors
  • 10 µg/ml Colcemid (Life Technologies)
  • 75 mM hypotonic KCl buffer (12.3 mM HEPES/0.53 mM EGTA/64.4 mM KCl; or commercially available from Stem Cell Technologies or Life Technologies), 37°C
  • 3:1 (v/v) methanol/glacial acetic acid fixative (Fisher; BDH)
  • 70%, 90%, 95%, and 100% ethanol
  • Phosphate buffered saline, pH 7.4 (PBS; without Ca2+ and Mg2+, Stem Cell Technologies; also see recipe)
  • 37% formaldehyde, reagent‐grade (BDH)
  • Pepsin (Sigma‐Aldrich)
  • 1 N HCl, pH 2.0
  • Hybridization mixture (see recipe)
  • Wash solution I (see recipe)
  • Wash solution II (see recipe)
  • Mounting medium containing antifade and DAPI (see recipe)
  • 25‐cm2 tissue culture flasks with vented blue plug seal caps (Becton Dickinson)
  • 15‐ and 50‐ml polypropylene conical tube (Becton Dickinson)
  • Precleaned microscope slides for hematology and histology (e.g., Micro World; http://www.mwrn.com/)
  • Lint‐free paper towels
  • Phase‐contrast microscope
  • 100‐ and 150‐ml glass slide staining jars and corresponding slide rack
  • Coverslips (22 × 60–mm; VWR)
  • Thick metal plate (∼1‐cm aluminum), e.g., 6 ×10–in.
  • 80°C oven
  • Molded plastic slide box for 25 slides (Fisher)
  • Parafilm
  • Platform shaker (Barnstaed/Thermolyne)
  • Light‐protected slide storage box
NOTE: All solutions and equipment coming into contact with live cells must be sterile, and aseptic techniques should be used accordingly. Furthermore, it is prudent to consider cultured and primary human cells as biohazardous and adhere to appropriate biohazard containment and disposal guidelines. Consult your institutional safety officer for specific instructions.NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO 2 to maintain pH 7.4.CAUTION: To avoid inhalation of fumes from glacial acetic acid, methanol, formaldehyde, and formamide solutions, it is recommended that cell fixation and formamide washes be performed in a fume hood. Formamide is a harmful and irritant organic solution. Wear suitable protective clothing and gloves when handling; avoid inhalation of fumes and skin contact. Use an excess of water in case of skin contact. Refer to Material Safety Data Sheet and consult your institutional guidelines for appropriate disposal of chemical waste.

Support Protocol 1: Image Capture

  Materials
  • Immersion oil for fluorescence and general microscopy (NA = 1.5150 ±0.0002, Cargille Laboratories)
  • Sample slide of labeled telomeres and chromosomes (see the protocol 1)
  • Reference slide of fluorescent beads (see protocol 4)
  • Fluorescence microscope (Zeiss Axioplan or equivalent) with the following components:
  •  Excitation and emission filters to match the probes used (DAPI/CY3 dichroic mirror/emission filter set, Chroma Technology)
  •  One high‐quality objective lens (e.g., Plan Apochromat 63×/1.4 Zeiss or equivalent)
  •  Stable fluorescent light source (e.g., 200 W hybrid mercury/xenon lamp, OptiQuip)
  •  Fine focusing adjustment in ≤0.2‐µm increments (e.g., piezo electric focus drive attached to the objective lens or fine focus control provided by the microscope)
  • Camera (e.g., MicroImager MI1400‐12 Q‐Imaging or equivalent) with the following characteristics:
  •  Variable exposure times with a maximum time of at least 10 sec to capture weak fluorescence signals
  •  At least 8 bits of photometric resolution (higher bit depth cameras generally are better as the upper 8 bits are less noisy and allow the user to select a sub‐range of interest from the full range)
  •  Small pixel size (∼7 × 7–µm such that better localization of the small telomere spots can be made)
  •  Large number of pixels (∼1000 × 1000 such that the entire metaphase spread is captured within the camera's field of view)
  •  No automatic gain control or the ability to disable this function (e.g., manual gain control)
  • Image acquisition software with the following features:
  •  Visualization of the captured image on a monitor
  •  Capture and storage of images in the proprietary “.IMG” format or the Windows Bitmap 8 bit/pixel “.BMP” format
  •  Histogram option to display the intensity variation in the image (this feature will give an indication of the values of the minimum and maximum pixel intensity values that are present in the image)
  •  Feature to store the raw image data from the camera (such that unmodified images captured under controlled conditions can be consistently and objectively analyzed later)
  •  Feature to capture and display images in continuous mode (optional)
  •  Feature to switch into “intensity enhancement” mode where low light levels are scaled to appear much brighter and is useful for focusing (optional)
  •  Feature to switch into “binning” mode, an acquisition mode where images are captured at a faster rate (e.g., lower exposure times) but at a lower spatial resolution and is useful for focusing (optional)
CAUTION: Ensure that all automatic gain features are turned off when capturing data for image analysis. This includes the gain control setting on the camera. It also includes the “intensity enhancement”, “binning” modes, and other similar modes that are available on various imaging systems.NOTE: Ensure that the immersion oil on the slide connects evenly with the objective as this will reduce the optical distortion of the captured and viewed images.

Support Protocol 2: Image Analysis

  Materials
  • Images of telomeres (see protocol 2)
  • Corresponding images of chromosomes (see protocol 2)
  • TFL‐Telo (telomere image analysis software, this software is available upon request from the authors: )
  • Pentium‐based personal computer with the following:
  •  Minimum of 8 Mb of RAM
  •  Graphics card with at least 256 colors and a display resolution of 640 × 480 pixels
  •  High resolution video monitor to match the video card
  •  Microsoft Windows 95, 98, or NT operating system

Support Protocol 3: System Calibration

  Materials
  • 0.2‐µm fluorospheres carboxylate‐modified microspheres, orange fluorescent (540/560 Molecular Probes)
  • 1:1 (v/v) recipePBS/FCS
  • Antifade reagent in glycerol/PBS (Slow Fade Antifade kit, component A, Molecular Probes)
  • PSXneo plasmids containing fixed numbers of telomere repeats (Hanish et al., ; Martens et al., )
  • 22 × 30–mm coverslips
  • Slide box
  • 1.5‐ml “sure lock” tubes (Rose Scientific)
  • Thermomixer (e.g., Eppendorf Model 5436) or 80°C water bath
  • Additional reagents and equipment for PNA probe hybridization (see protocol 1), image capture (see protocol 2), and image analysis (see protocol 3)
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Figures

Videos

Literature Cited

Literature Cited
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   Blackburn, E.H. 1994. Telomeres: No end in sight Cell 77:621‐623.
   Blasco, M.A., Lee, H.‐W., Hande, M.P., Samper, E., Lansdorp, P.M., DePinho, R.A. and Greider, C.W. 1997. Telomere shortening and tumor formation by mouse cells lacking telomerase RNA Cell 91:25‐34.
   d'Adda di Fagagna, F., Hande, M.P., Tong, W.‐M., Lansdorp, P.M., Wang, Z.‐Q. and Jackson, S.P. 1999. Functions of poly (ADP‐ribose) polymerase in controlling telomere length and chromosomal stability (letter). Nature Genet. 23:76‐80.
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