Preparation of Single Cells from Solid Tissues for Analysis by PCR

N.M. Sawtell1

1 Children's Hospital Medical Center, Cincinnati, Ohio
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 25A.2
DOI:  10.1002/0471142727.mb25a02s58
Online Posting Date:  May, 2002
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Abstract

This unit details a protocol for the separation of solid tissues into single cell suspensions for subsequent analysis of nucleic acids and protein.Tissues are fixed in situ by perfusion, which terminates cell processes and thus changes that would accompany dissociating the living tissue. The procedure is particularly useful when the cell type of interest represents a minor population relative to other cells types in the tissue. Once separated, individual cells or groups of a particular cell type can then be analyzed using PCR strategies. The procedure can also be adapted to allow the quantification of the number of cells within a tissue containing specific nucleic acid sequences, for example, a particular viral DNA or RNA sequence.

     
 
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Table of Contents

  • Basic Protocol 1: Perfusion Fixation and Enrichment of Single Cells
  • Support Protocol 1: Determining Number of Neurons Recovered
  • Alternate Protocol 1: Nonperfusion Fixation with STF Solution
  • Alternate Protocol 2: Preparation of lacZ‐Expressing Cells from Solid Tissues
  • Basic Protocol 2: Analysis of Single Cells by PCR
  • Basic Protocol 3: Analysis of Enriched Cell Populations by RT‐PCR
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Perfusion Fixation and Enrichment of Single Cells

  Materials
  • Streck tissue fixative (STF; Streck Laboratories)
  • Animal (e.g., mouse)
  • Sodium pentobarbital
  • 95% ethanol
  • 0.25% (w/v) collagenase CLS I (Worthington) in recipeHank's balanced salt solution (HBSS; see recipe)
  • Triple 0.2‐µm filtered nanopure (3×F) H 2O
  • Percoll (Pharmacia): adjust to pH 6.0 with HCl
  • Peristaltic pump (BRL CP‐600 or equivalent) and appropriate tubing
  • 15‐ and 50‐ml conical tubes
  • 27‐G needle
  • 80°C water bath
  • Dissecting microscope (optional)
  • Clean dissection tools (e.g., forceps, scalpel blades, hemostat, 25‐G needles)
  • Glass slides: bake overnight (3 hr minimum) at 250°C
  • 200‐ and 1000‐µl aerosol‐resistant pipette tips
  • 15‐ml polystyrene conical tubes
  • 9‐in. Pasteur pipettes: bake overnight (3 hr minimum) at 250°C
  • Additional reagents and equipment for determining number of neurons recovered (see protocol 2Support Protocol), and analyzing DNA or RNA from single‐cell populations (see Support Protocols protocol 52 and protocol 63)
NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) or must conform to governmental regulations regarding the care and use of laboratory animals.NOTE: Depending upon the final application of the cells, all materials must be DNase‐ and RNase‐free, and free of contaminating nucleic acids which could interfere with the interpretation of downstream PCR.

Support Protocol 1: Determining Number of Neurons Recovered

  Materials
  • Cell pellet (see protocol 1)
  • recipeCresyl violet solution (see recipe)
  • 95% and 100% ethanol
  • Xylene
  • Permount
  • Superfrost/Plus glass slides (Fisher) or equivalent with coverslips
  • Additional reagents and equipment for analyzing neuron‐specific proteins (e.g., neurofilament 200 kDa peptide) by immunohistochemistry (Sawtell, )

Alternate Protocol 1: Nonperfusion Fixation with STF Solution

  • Harvested tissue, fresh
  • recipeHBSS (see recipe)

Alternate Protocol 2: Preparation of lacZ‐Expressing Cells from Solid Tissues

  • Glutaraldehyde
  • 100 µg/ml Xgal in recipeXgal buffer (see recipe)

Basic Protocol 2: Analysis of Single Cells by PCR

  Materials
  • Enriched cell sample (see protocol 1 or Alternate Protocols protocol 31 or protocol 42)
  • Triple 0.2‐µm filtered nanopure (3×F) H 2O
  • recipePonceau S solution (see recipe)
  • Immobilized‐DNase on PVP beads (Mobitec)
  • recipeDNase reaction buffer (see recipe)
  • recipePCR/PK solution (see recipe)
  • DNA standards—e.g., cloned segments of HSV genome containing the gene being amplified (e.g., thymidine kinase)
  • recipePCR amplification solution (see recipe)
  • Taq DNA polymerase (Life Technologies)
  • 200‐µl PCR tubes
  • Dissecting microscope
  • PCR Gene Amp 2400 (Perkin Elmer Cetus)
  • Gene screen plus nylon membrane (NEN Life Science Products)
  • Storage phosphor screen (Molecular Dynamics)
  • Imagequant software
  • Additional reagents and equipment for quantitating standards (unit 15.7; Sawtell and Thomson, ), PCR (unit 15.1), nondenaturing polyacrylamide gel electrophoresis (units 2.5 & 2.7), UV‐crosslinking DNA to filters (unit 2.9), hybridizing blots with oligonucleotides (units 2.9 & 6.4), labeling oligonucleotides (units 4.6, 4.8 & 15.7), and phosphorimaging ( 3.NaN)

Basic Protocol 3: Analysis of Enriched Cell Populations by RT‐PCR

  Materials
  • recipeProteinase K solution (see recipe)
  • 40 mM PMSF, fresh
  • RNase‐free DNase I: 3 U RNase‐free DNase I (Boehringer Mannheim)/25 mM DTT/ 2.5 U placental RNase inhibitor
  • 8 pmols/µl reverse transcriptase primer
  • recipeReverse‐transcription reaction mix (see recipe)
  • 200 U/µl SuperScript II reverse transcriptase (Life Technologies)
  • recipePCR amplification solution (see recipe)
  • 1.25 U Taq DNA polymerase (Life Technologies)
  • PCR tubes
  • Additional reagents and equipment for obtaining dissociated perfusion‐fixed cells (see protocol 1) and PCR optimization (unit 15.1)
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Figures

Videos

Literature Cited

   Coligan, J.E., Kruisbeek, A.M., Margulies, D.H., Shevach, E.M., and Strober, W. (eds.) 2001. Current Protocols in Immunology. John Wiley & Sons New York.
   Gilbert, S. 1994. Developmental Biology 4th ed. Sinauer Associates, Inc. Sunderland, Mass.
   Katz, J.P., Bodin, E.T., and Coen, D.M. 1990. Quantitative polymerase chain reaction analysis of herpes simplex virus DNA in ganglia of mice infected with replication‐incompetent mutants. J. Virol. 64:4288‐4295.
   Mullis, K.B. and Falona, F.A. 1987. Specific synthesis of DNA in vitro via a polymerase‐catalyzed chain reaction. Meth. Enzymol. 155:335‐350.
   Pretlow II, T.G. and Pretlow, T.P. (eds.) 1982. Cell Separation. Methods and Selected Applications. Academic Press, New York.
   Sawtell, N.M. 1997. Comprehensive quantification of herpes simplex virus latency at the single cell level. J Virol. 71:5423‐5431.
   Sawtell, N.M. and Thomson, R.L. 1992. Herpes simplex virus type 1 latency‐associated transcription unit promotes anatomical site‐dependent establishment and reactivation from latency. J. Virol 66:2157‐2169.
   Sawtell, N.M., Poon, D.K., Tansky, C.S., and Thompson, R.L. 1998. The latent HSV‐1 genome copy number in individual neurons is virus strain specific and correlates with reactivation. J. Virol. 72:5343‐5350.
   Sawtell, N.M., Thompson, R.L., Stanberry, L.R. and Bernstein, D.I. 2001. Early intervention with high‐dose acyclovir treatment during primary herpes simplex virus infection reduces latency and subsequent reactivation in the nervous system in vivo. J.I.D. 184:964‐971.
   Thompson, R.L. and Sawtell, N.M. 1997. The herpes simplex virus type 1 latency‐associated transcript gene regulates the establishment of latency. J. Virol. 71:5432‐5440.
   Thompson, R.L. and Sawtell, N.M. 2000. Replication of herpes simplex virus type 1 within the trigeminal ganglia is required for high frequency but not high viral genome copy number latency. J Virol. 74:965‐974.
   Thompson, R.L. and Sawtell, N.M. 2001. Herpes simplex type 1 latency‐associated transcript gene promotes neuronal survival. J Virol. 75:6660‐6675.
   Virchow, R. 1863. Cellular pathology: as based upon physiological and pathological histology. 2nd ed. translated by F. Chance, J.B. Lippincott Philadelphia.
Key References
   Sawtell, N.M. 1997. See above.
  This manuscript describes the procedure as used to quantify viral latency and includes several critical validation experiments.
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