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Gene Expression Analysis of a Single or Few Cells

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

¹Ludwig‐Maximilians‐University of Munich, Munich, Germany

Publication Name:

Current Protocols in Human Genetics

Unit Number:

Unit 11.8

DOI:

10.1002/0471142905.hg1108s44

Online Posting Date:

February, 2005

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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 semi-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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Unit Introduction
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: Nonradioactive 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 2D)
5× RT buffer (Life Technologies)
0.1 M DTT (Life Technologies)
10% (v/v) Igepal
cDNA synthesis primers:
- For mRNA amplification for ³100 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)
Igepal wash buffer (see recipe)
Tween 20 wash buffer (see recipe)
40 mM MgCl₂
2 mM dGTP
200 mM KH₂PO₄
Tailing 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 MgCl₂)
- 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 -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-1: 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 2.7)

Alternate Protocol 1: Extraction of mRNA from Small Tissue Biopsies

Additional Materials (also see Basic Protocol 1)

Biopsy sample, 1 to 1.5 mm in diameter
Liquid nitrogen
Mortar and pestle
UV transilluminator
Dry ice

Alternate Protocol 2: Extraction of mRNA from Microdissected Samples

Materials (also see Basic Protocol 1)

Resectioned tissue snap-frozen in liquid nitrogen and stored at –80°C (see Alternate Protocol 1)
OCT embedding compound (Tissue-Tek, Miles)
Mayer's hematoxylin solution (Sigma)
70%, 95%, and 100% ethanol
Lysis buffer from Oligo dT kit (see Basic Protocol 1)

Cryostat
Slides for the PALM Laser-MicroBeam System (PALM)
PALM Laser-MicroBeam System (PALM)

Basic Protocol 2: Nonradioactive Gene Expression Analysis on Nylon Arrays

Materials

Expand Long Template (ELT) PCR system (Roche Diagnostics) including:
- 10× ELT buffer 1 (17.5 mM MgCl₂)
- 3.5 U/µl DNA polymerase mix
1/7 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
Development buffer 1 (see recipe)
Development 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)
Development 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

Figure 11.8.1

Global amplification of mRNA from a few or single cells. mRNA is captured by paramagnetic beads (1), and primed using random and oligo dT primers containing a poly C flanking region (2). cDNA synthesis starts from both primers (3; CFL5c8 is omitted in 3 and 4). After RNA removal, a poly G tail is added by TdT. Using the poly C containing CP2 primer, all sequences can be amplified (5).

View Image
Figure 11.8.2

Isolation of small tissue samples by laser microdissection and catapulting using the PALM system.

View Image

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.
	Frost, A.R., Eltoum, I.-E., and Siegal, G.P. 2001. Laser Capture Microdissection. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidmon, J.A. Smith, and K. Struhl, eds.) pp. 25A.1.1-25A.1.24. John Wiley & Sons, Hoboken, N.J.
	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.