Analysis of mRNA Populations from Single Live and Fixed Cells of the Central Nervous System

James Eberwine1, Peter Crino1

1 University of Pennsylvania Medical Center, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 5.3
DOI:  10.1002/0471142301.ns0503s00
Online Posting Date:  May, 2001
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This unit presents a method for the amplification of poly(A)+ mRNA extracted from the cytoplasm of a single cell. After cDNA is synthesized from the mRNA, it is made double stranded, denatured, and reverse transcribed to yield antisense RNA (aRNA). Another round of amplification results in a relatively large amount of aRNAs in essentially the same proportion as in the starting mRNA population. RNA amplification protocols can be used for many purposes, including generation of disease expression profiles, making of cDNA libraries, and generation of diagnostics and therapeutics for disease. An alternate protocol is used to amplify RNAs from single neurons in fixed tissue specimens. Support protocols gives instructions for reverse northern analysis, which allows analysis of the presence or absence and relative levels of mRNA expression in selected cells, and a convenient method to assess the RNA content in fixed tissue sections using the fluorescent dye acridine orange (which binds singleā€stranded nucleic acids).

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Table of Contents

  • Basic Protocol 1: Single‐Cell mRNA Amplification from Live Cells
  • Alternate Protocol 1: Single‐Cell mRNA Amplification from Immunostained Fixed Cells
  • Support Protocol 1: Reverse Northern Analysis of mRNA
  • Support Protocol 2: Acridine Orange Labeling
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Single‐Cell mRNA Amplification from Live Cells

  • Electrode solution with T7‐oligo(dT) 24 primer (see recipe)
  • Cells
  • 10× electrode buffer (see recipe), adjusted to pH 8.3 with 2 M NaOH
  • 2.5 mM 4dNTP mix
  • 20 U/µl avian myeloblastosis virus reverse transcriptase (AMV‐RT; Seikagaku America)
  • 5 M NaCl
  • 100% ethanol
  • E. coli tRNA
  • 10× second‐strand buffer (see recipe)
  • 1 U/µl T4 DNA polymerase
  • 1 U/µl Klenow fragment of E. coli DNA polymerase
  • 10× S1 buffer (see recipe)
  • 1 U/µl S1 nuclease (Boehringer Mannheim) in 1× S1 buffer
  • 1:1 (v/v) buffered phenol ( appendix 2A)/chloroform
  • 10× Klenow filling in (KFI) buffer (see recipe)
  • 10 mM each dATP, dCTP, dGTP, and dTTP
  • TE buffer, RNase‐free (e.g., made with DEPC‐treated water; appendix 2A)
  • 10× RNA amplification buffer (see recipe)
  • 100 mM dithiothreitol (DTT)
  • 2.5 mM 4NTP mix
  • 2.5 mM 3NTP mix (A, G, U)
  • 0.1 mM CTP
  • 10 mCi/ml [α‐32P]CTP (800 Ci/mmol)
  • 0.1 U/µl RNasin (Promega) or equivalent RNase inhibitor
  • 2000 U/µl T7 RNA polymerase (Epicentre Technologies)
  • 3 M sodium acetate, pH 6.5
  • Random hexanucleotide primers
  • 10× RT buffer (see recipe)
  • 100 ng/µl T7‐oligo(dT) 24 (for T7 sequence, see recipe for electrode buffer with primer)
  • Diethylpyrocarbonate (DEPC)‐treated water ( appendix 2A)
  • Formaldehyde
  • 10× MOPS (see recipe)
  • Formamide
  • Gel loading dye ( appendix 2A)
  • 10% (w/v) trichloroacetic acid (TCA)
  • Electrode apparatus (see unit 6.3)
  • 1‐ml sterile syringe
  • 14°, 37° to 42°, 75°, 85°, and 95°C heating blocks or water baths
  • Dry ice/ethanol bath
  • 0.025‐µm Millipore filter
  • Additional reagents and equipment for separating RNAs by agarose gel electrophoresis (CPMB UNIT and appendix 3A in this manual)

Alternate Protocol 1: Single‐Cell mRNA Amplification from Immunostained Fixed Cells

  • Neural tissue
  • 4% (w/v) paraformaldehyde or 70% ethanol/150 mM NaCl
  • 0.5×, 2×, and 5× SSC ( appendix 2A)
  • IST reaction buffer (see recipe)
  • 0.5 U/µl avian myeloblastosis virus reverse transcriptase (AMV‐RT; Seikagaku America)
  • cDNA synthesis buffer (see recipe)
  • Microelectrode solution (see recipe)
  • Paraffin
  • Rubber cement
  • Poly‐L‐lysine–coated glass slides ( appendix 2A)
  • Dissecting microscope
  • Additional reagents and equipment for sectioning and mounting tissues (unit 1.1) and immunolabeling tissue sections (unit 1.2)

Support Protocol 1: Reverse Northern Analysis of mRNA

  • Fixed tissue sections (see protocol 2)
  • Citric acid/sodium phosphate buffer (CPBS buffer; see recipe)
  • Acridine orange staining solution (see recipe)
  • Fluorescence microscope (see unit 2.1)
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Literature Cited

Literature Cited
   Bargas, J., Howe, A., Eberwine, J., and Surmeier, J. 1994. Acutely isolated rat neostriatal neurons express four types of high‐voltage activated calcium currents. J. Neurosci. 14:6667‐6686.
   Cao, Y., Wilcox, K., Martin, C., Eberwine, J., and Dichter, M. 1996. Presence of mRNA for glutamic acid decarboxylase in both excitatory and inhibitory neurons: Implications for a new form of neuronal plasticity. Proc. Natl. Acad. Sci. U.S.A. 93:9844‐9849.
   Chee, M., Yang, R., Hubbell, E., Berno, A., Huang, X., Stern, D., Winkler, J., Lockhart, D., Morris, M., and Fodor, S. 1996. Accessing genetic information with high‐density DNA arrays. Science 274:610‐614.
   Crino, P. and Eberwine, J. 1996. Molecular characterization of the dendritic growth cone: Regulated mRNA transport and local protein synthesis. Neuron 17:1173‐1187.
   Crino, P., Dichter, M., Trojanowski, J., and Eberwine, J. 1996. Single cell molecular pathology: An analysis of tuberous sclerosis. Proc. Natl. Acad. Sci. U.S.A. 93:14152‐14157.
   Eberwine, J. 1996. Amplification of mRNA populations using aRNA generated from immobilized oligo‐dT‐T7 primed cDNA. BioTechniques 20:584‐586.
   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.
   Eberwine, J., Crino, P., and Dichter, M. 1995. Single cell mRNA amplification: Basic science and clinical implications. Neuroscientist 1:200‐211.
   Finnell, R., VanWaes, M., Bennett, G., and Eberwine, J. 1993. Lack of concordance between heat shock proteins and the development of tolerance to teratogen induced neural tube defects. Dev. Genet. 14:137‐147.
   Lockhart, D., Dong, H., Byrne, M., Follettie, M., Gallo, M., Chee, M., Mittmann, M., Wang, C., Kobayashi, M., Horton, H., and Brown, E. 1996. Expression monitoring by hybridization to high‐density oligonucleotide arrays. Nature Biotech. 14:1675‐1680.
   Mackler, S. and Eberwine, J. 1993. Diversity of glutamate receptor subunit mRNA expression within live hippocampal CA1 neurons. Mol. Pharmacol. 44:308‐315.
   Mackler, S., Brooks, B., and Eberwine, J. 1992. Stimulus‐induced coordinate changes in mRNA abundance in single post‐synaptic hippocampal CA1 neurons. Neuron 9:539‐548.
   Miyashiro, K., Dichter, M., and Eberwine, J. 1994. On the nature and distribution of mRNAs in hippocampal neurites: Implications for neuronal functioning. Proc. Natl. Acad. Sci. U.S.A. 91:10800‐10804.
   Nair, S. and Eberwine, J. 1994. Molecular correlates of corticosterone action in hippocampal subregions. Neurobiology of steroids. In Methods in Neurosciences, Vol. 22 (P. Conn, series ed.) pp.314‐329. Academic Press, New York.
   Surmeier, J., Eberwine, J., Wilson, C., Cao, Y., Stefani, A., and Kitai, S. 1992. Cellular and molecular evidence for the co‐localization of dopamine receptor subtypes in acutely‐isolated rat striatonigral neurons. Proc. Natl. Acad. Sci. U.S.A. 89:10178‐10182.
   Tecott, L., Barchas, J., and Eberwine, J. 1988. In situ transcription: Specific synthesis of cDNA in fixed tissue sections. Science 240:1661‐1664.
   Topaloglu, H. and Sarnat, H. 1989. Acridine orange‐RNA fluorescence of maturing neurons in the perinatal rat brain. Anat. Rec. 224:88‐93.
   van Gelder, R., von Zastrow, M., Yool, A., Dement, W., Barchas, J., and Eberwine, J. 1990. Amplified RNA (aRNA) synthesized from limited quantities of heterogeneous cDNA. Proc. Natl. Acad. Sci. U.S.A. 87:1663‐1667.
   Zangger, I., Tecott, L., and Eberwine, J. 1989. In situ transcription: A methodological study using propiomelanocortin gene expression in the rat pituitary as a model. Technique 1:108‐117.
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