Gene Expression Analysis of a Single or Few Cells

Christoph A. Klein1, Dietlind Zohlnhöfer1, Karina Petat‐Dutter1, Nicole Wendler1

1 Ludwig‐Maximilians‐University of Munich, Munich, Germany
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 25B.8
DOI:  10.1002/0471142727.mb25b08s61
Online Posting Date:  February, 2003
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Abstract

The need to analyze rare cells is based on the nature of tissue differentiation and regeneration, the initiation and propagation of disease processes in multicellular organisms, and the functional diversity of individual cells. Gene transcription is the most important regulatory mechanism by which a phenotype and functional state of a cell is determined. Therefore, procedures for the qualitative and quantitative assessment of mRNA abundance are important. This unit presents a protocol for semiā€quantitative analysis of gene expression of a single cell and quantitative representation of expressed genes from >10 to 30 cells. A basic protocol for array hybridization on nylon filters is provided because such filters are available in every laboratory. Tissue samples contain many different cell types in variable amounts, so their analysis may require microdissection; a protocol for obtaining cryosections is given. Finally, a simple procedure to prepare the data for statistical analysis is also provided.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Global Amplification of Single‐Cell cDNA
  • Alternate Protocol 1: Extraction of mRNA from Small Tissue Biopsies
  • Alternate Protocol 2: Extraction of mRNA from Microdissected Samples
  • Basic Protocol 2: Non‐Radioactive Gene Expression Analysis on Nylon Arrays
  • Basic Protocol 3: Data Analysis of Hybridized cDNA Arrays
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Global Amplification of Single‐Cell cDNA

  Materials
  • Oligo dT kit (Dynal) including:
  •  Dynabeads Oligo (dT) 25
  •  Washing buffer containing LiDS
  •  Lysis buffer
  • Phosphate‐buffered saline (PBS; appendix 22)
  • 5× RT buffer (Life Technologies)
  • 0.1 M DTT (Life Technologies)
  • 10% (v/v) Igepal
  • cDNA synthesis primers:
  • For mRNA amplification for ≥100 cells:
  •  CFL5C6: 5′‐(CCC) 5 GTC TAG ANN NNN N‐3′ (200 µM)
  • For single cells and 5′ and 3′ coverage:
  •  CFl5C8: 5′‐(CCC) 5 GTC TAG ANN NNN NNN‐3′ (200 µM)
  •  CFl5CT: 5′‐(CCC) 5 GTC TAG ATT TTT TTT TTT TTT TVN‐3′ (100 µM)
  •  CFL5 primer mix: 1 vol CFl5c8 (200 µM) + 1 vol CFl5cT (100 µM)
  • For the use of 3′‐restricted oligo arrays:
  •  CFl5CT (24): 5′‐(CCC) 5 GTC TAG ATT (T) 22VN‐3′
  • 10 mM and 200 µM dNTPs
  • Reverse transcriptase (Superscript II; Life Technologies)
  • recipeIgepal wash buffer (see recipe)
  • recipeTween 20 wash buffer (see recipe)
  • 40 mM MgCl 2
  • 2 mM dGTP
  • 200 mM KH 2PO 4
  • recipeTailing wash buffer (see recipe)
  • Mineral oil
  • Terminal deoxynucleotide transferase (TdT; Amersham Pharmacia Biotech)
  • Expand Long Template (ELT) PCR system (Roche Diagnostics) including:
  •  10× ELT buffer 1 (17.5 mM MgCl 2)
  •  3.5 U/µl DNA polymerase mix
  • 20% (v/v) formamide
  • PCR primer, CP2: 5′‐ TCA‐GAA‐TTC‐ATG‐CCC‐CCC‐CCC‐CCC‐CCC‐3′ (24 µM)
  • 1× PCR buffer (Sigma)
  • Primers for β‐actin: 5′‐ CTA CGT CGC CCT GGA CTT CGA GC‐3′ and 5′‐GAT GGA GCC GCC GAT CCA CAC GG‐3′
  • Primers for EF‐1α: 5′‐ GCA GTG CAC ACA CAG AGG TGT A‐3′ and 5′‐ CTA CCG CTA GGA GGC TGA GCA A‐3′
  • 0.75 U Taq DNA polymerase (Sigma)
  • Magnet separation apparatus for 0.2‐ml tubes (Dynal)
  • 0.2‐ml PCR tubes
  • 15‐ to 50‐ml tubes
  • Roller‐bottle apparatus or other rotisserie‐type rotator
  • Thermal cycler
  • Hybridization oven or other rotator with temperature control
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.5 )

Alternate Protocol 1: Extraction of mRNA from Small Tissue Biopsies

  • Biopsy sample
  • Liquid nitrogen
  • Dry ice
  • Mortar and pestle

Alternate Protocol 2: Extraction of mRNA from Microdissected Samples

  • Resectioned tissue snap‐frozen in liquid nitrogen and stored at −80°C (see protocol 2)
  • OCT embedding compound (Tissue‐Tek, Miles; also see unit 25.1)
  • Mayer's hematoxylin solution (Sigma)
  • 70%, 95%, and 100% ethanol
  • Lysis buffer from Oligo dT kit (see protocol 1)
  • Cryostat
  • Slides for the PALM Laser‐MicroBeam System (PALM)
  • PALM Laser‐MicroBeam System (PALM)

Basic Protocol 2: Non‐Radioactive Gene Expression Analysis on Nylon Arrays

  Materials
  • Expand Long Template (ELT) PCR system (Roche Diagnostics) including:
  •  10× ELT buffer 1 (17.5 mM MgCl 2)
  •  3.5 U/µl DNA polymerase mix
  • recipe1/7 dNTP mix (see recipe)
  • 20% formamide
  • CP2 primer: 5′‐ TCA‐GAA‐TTC‐ATG‐CCC‐CCC‐CCC‐CCC‐CCC‐3′ (24 µM)
  • Digoxigenin‐11‐dUTP (Dig‐UTP), alkali labile (Roche Diagnostics)
  • Sample
  • DIG Easy Hyb solution (Roche Diagnostics)
  • E.coli DNA
  • DNase I
  • Labeled probe
  • Herring sperm DNA (Invitrogen)
  • 20× SSC
  • 10% SDS
  • recipeDevelopment buffer 1 (see recipe)
  • recipeDevelopment buffer 2 (see recipe)
  • DIG Luminescent Detection Kit (Roche Diagnostics) containing:
  •  Blocking reagent
  •  750 U/ml anti‐digoxigenin‐AP (Fab fragment) antibody
  •  11.6 mg/ml CSPD
  • Tween 20 (Sigma)
  • recipeDevelopment buffer 3 (see recipe)
  • Thermal cycler
  • Nylon membrane containing an array of cDNAs (either self‐prepared or commercially available)
  • Hybridization tubes
  • Hybridization oven or other rotator with temperature control
  • 1.5‐ml microcentrifuge tubes
  • Acetate sheets
  • Whatman 3MM filter paper
  • Biomax ML film (Kodak)

Basic Protocol 3: Data Analysis of Hybridized cDNA Arrays

  Materials
  • Photographic step tablet (Kodak)
  • Transparency scanner that can be calibrated (e.g., SNAPSCAN, Agfa)
  • Labscan software or equivalent (Scanwise v. 1.2.1, Agfa)
  • Array Vision software or equivalent (Clontech)
  • Excel software or equivalent (Microsoft)
  • SPSS software or equivalent (SPSS)
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Figures

Videos

Literature Cited

Literature Cited
   Belyavsky, A., Vinogradova, T., and Rajewsky, K. 1989. PCR‐based cDNA library construction: General cDNA libraries at the level of a few cells. Nucleic Acids Res. 17:2919‐2932.
   Brady, G. 2000. Expression profiling of single mammalian cells–small is beautiful. Yeast 17:211‐217.
   Brady, G. and Iscove, N.N. 1993. Construction of cDNA libraries from single cells. Methods Enzymol. 225:611‐623.
   Brail, L.H., Jang, A., Billia, F., Iscove, N.N., Klamut, H.J., and Hill, R.P. 1999. Gene expression in individual cells: Analysis using global single cell reverse transcription polymerase chain reaction (GSC RT‐PCR). Mutat.‐Res. 406:45‐54.
   Eberwine, J., Yeh, H., Miyashiro, K., Cao, Y., Nair, S., Finnell, R., Zettel, M., and Coleman, P. 1992. Analysis of gene expression in single live neurons. Proc. Natl. Acad. Sci. U.S.A. 89:3010‐3014.
   Kacharmina, J.E., Crino, P.B., and Eberwine, J. 1999. Preparation of cDNA from single cells and subcellular regions. Methods Enzymol. 303:3‐18.
   Klein, C.A., Seidl, S., Petat‐Dutter, K., Offner, S., Geigl, J.B., Schmidt‐Kittler, O., Wendler, N., Passlick, B., Huber, R.M., Schlimok, G., Baeuerle, P.A., and Riethmuller, G. 2002. Combined transcriptome and genome analysis of single micrometastatic cells. Nat. Biotechnol. 20:387‐392.
   Luo, L., Salunga, R.C., Guo, H., Bittner, A., Joy, K.C., Galindo, J.E., Xiao, H., Rogers, K.E., Wan, J.S., Jackson, M.R., and Erlander, M.G. 1999. Gene expression profiles of laser‐captured adjacent neuronal subtypes. Nat. Med. 5:117‐122.
   Zohlnhofer, D., Richter, T., Neumann, F., Nuhrenberg, T., Wessely, R., Brandl, R., Murr, A., Klein, C.A., and Baeuerle, P.A. 2001a. Transcriptome analysis reveals a role of interferon‐gamma in human neointima formation. Mol. Cell. 7:1059‐1069.
   Zohlnhofer, D., Klein, C.A., Richter, T., Brandl, R., Murr, A., Nuhrenberg, T., Schomig, A., Baeuerle, P.A., and Neumann, F.J. 2001b. Gene expression profiling of human stent‐induced neointima by cDNA array analysis of microscopic specimens retrieved by helix cutter atherectomy: Detection of FK506‐binding protein 12 upregulation. Circulation. 103:1396‐1402.
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