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

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

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 single?stranded nucleic acids).

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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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Materials

Basic Protocol 1: Single‐Cell mRNA Amplification from Live Cells

Materials

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‐³² P]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

Materials

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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Figures

Figure 5.3.1 Schematic of mRNA harvesting from a single cell. A patch pipet is used to sample the cytoplasmic RNA from a single cell in dispersed cell cultures, slice cultures, or fixed tissue sections.

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Figure 5.3.2 aRNA amplification schematic. The individual steps in the aRNA protocol are correlated with the molecular biology product resulting from the completion of each set of steps.

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Figure 5.3.3 Nestin‐immunolabeled cortical section from patient with tuberous sclerosis and epilepsy. Numerous nestin‐labeled abnormal neurons are seen. Arrow points to space where single cell was microdissected and aspirated into a recording electrode. The RNA amplified from this single cell can be used as a probe in expression profiling or used as a source for PCR amplification of individual mRNA species, differential display of subpopulations of the mRNA, or to make cDNA libraries. Nestin antibody (AN129, diluted 1:1000 for these studies) was generously provided by Dr. J. Trojanowski, Univ. of Pennsylvania.

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Figure 5.3.4 Reverse northern (slot) blot probed with ³² P‐labeled aRNA from a single immunolabeled human neuron. Brain specimens were obtained intraoperatively from a patient with tuberous sclerosis (an autosomal form of cerebral cortical dysplasia) and epilepsy. Brain samples were immersion‐fixed in 4% paraformaldehyde,embedded in paraffin, and cut at 7 µm. Sections were probed with anti‐nestin (human) antibodies. Blots were generated using linearized plasmid cDNAs as a target by binding these cDNAs to nylon membrane (Hybond), hybridizing with the aRNA probe, washing in SSC solutions containing 0.1% SDS, air drying, and apposing the probed membrane to film. The autoradiographic intensity of each hybridizing plasmid on the autoradiogram is a direct reflection of the amount of aRNA in the probe, which in turn reflects the amount of that particular mRNA in the original cell. A1, α3 subunit of the GABA‐A receptor; A2, microtubule‐associated protein 2 (MAP2); A3, α‐subunit of calcium/calmodulin kinase II; A4, α‐internexin (courtesy R. Liem); B1, connexin 26 (courtesy D. Paul); B2, connexin 32; B3, human nestin (courtesy U. Lendahl); B4, low molecular weight neurofilament (courtesy V. Lee); C1, GAP43; C2, high molecular weight neurofilament; C3, Ki67 (ATCC); C4, α2 subunit of the GABA A receptor; D1, pBluescript plasmid cDNA.

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Literature Cited

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