Table Of ContentPreface
Genomic sequences, now emerging at a rapid rate, are greatly expediting
certain aspects of molecular biology. However, in more complex organisms,
predicting mRNA structure from genomic sequences can often be difficult.
Alternative splicing, the use of alternative promoters, and orphan genes
without known analogues can all offer difficulties in the predictions of the
structure of mRNAs or even in gene detection. Both computational and
experimental methods remain useful for recognizing genes and transcript
templates, even in sequenced DNA. Methods for producing full-length
cDNAs are important for determining the structure of the proteins the
mRNA encodes, the position of promoters, and the considerable regulatory
information for translation that may be encoded in the 5' untranslated
regions of the mRNA.
Methods for studying levels of mRNA and their changes in different
physiological circumstances are rapidly evolving, and the information from
this area will rival the superabundance of information derived from genomic
sequences. In particular, cDNAs can be prepared even from single cells,
and this approach has already yielded valuable information in several areas.
To the extent that reliable and reproducible information, both quantitative
and qualitative, can be generated from very small numbers of cells, there are
rather remarkable possibilities for complementing functional and genetic
analysis of developmental patterns with descriptions of changes in mRNAs.
Dense array analysis promises to be particularly valuable for the rapid
expression pattern of known genes, while other methods such as gel display
approaches offer the opportunity of discovering unidentified genes or for
investigating species whose cDNAs or genomes have not been studied inten-
sively.
Knowledge of mRNA structure, genomic location, and patterns of ex-
pression must be converted into information of the function of the encoded
proteins. Each gene can become the subject of years of intensive study.
Nevertheless, a number of methods are being developed that use cDNA
to predict properties or permit the selective isolation of cDNAs encoding
proteins with certain general properties such as subcellular location. This
volume presents an update of a number of approaches relevant to the areas
referred to above. The technology in this field is rapidly evolving and these
contributions represent a "snapshot in time" of the number of currently
available and useful approaches to the problems referred to above.
SHERMAN M. WEISSMAN
xiii
Contributors to Volume 303
Article numbers are in parentheses following the names of contributors.
Affiliations listed are current.
CHRISTOPHER ASTON (4), Department of LISA A. COLLINS-RAcIE (26), Genetics Insti-
,yrtsimehC .W .M Keck Laboratory for Bio- ,etut ,egdirbmaC sttesuhcassaM 04120
molecular Imaging, New York ,ytisrevinU PETER B. CRINO (1), Department of Neurol-
New York, New York 30001 ogy, University of Pennsylvania Medical
NAMADEV BASKARAN (3, 16), Genome -arehT ,retneC ,aihpledalihP Pennsylvania 40191
peutics Corporation, Waltham, -uhcassaM JOHAN T. NED DUNNEN (7), Department of
setts 35420 Human ,sciteneG Leiden University Medi-
ALASTAIR J. H. BROWN (22), Trafford ertneC lac ,retneC 3332 Al Leiden, ehT sdnalrehteN
for Medical ,hcraeseR ytisrevinU of ,xessuS LUDA DIATCHENKO (20), CLONTECH
Brighton, BN1 9RY, United Kingdom
,seirotarobaL Inc., Palo Alto, California
PATRICK O. BROWN (12), Department of Bio- 0324-30349
,yrtsimehc Stanford University School of
RADOJE DRMANAC (11), Hyseq, Inc., Sunny-
enicideM and Howard Hughes Medical In-
,elav California 68049
,etutits Stanford, California 8245-50349
SNEZANA DRMANAC (11), Hyseq, Inc., Sunny-
ALAN J. BUCKLER (6), Axys ,slacituecamrahP
,elav California 68049
La Jolla, California 73029
McKEOUGH DUCKETT (26), sciteneG ,etutitsnI
JULIAN F. BURKE (22), School of Biological
,egdirbmaC sttesuhcassaM 04120
,secneicS University of Sussex, Brighton,
JAMES EBERWINE (1), Departments of -rahP
BN1 9RY, United Kingdom
macology and Psychiatry, University of
KONRAD WOSS3(B (13), Abt. Lehrach, Max
ainavlysnneP Medical ,retneC ,aihpledalihP
Planck Institut far Molekulare Genetik,
Pennsylvania 40191
Berlin (Dahlem) ,59141-D Germany
MICHAEL B. EISEN (12), Departments of -eG
DOLORES J. CAHILL (13), Abt. Lehrach, Max
sciten and Biochemistry, Stanford -revinU
Planck Institut far Molekulare Genetik,
sity School of ,enicideM Stanford, -rofilaC
Berlin (Dahlem) ,59141-D Germany
ain 8245-50349
PIERO CARNINCI (2), Laboratory for Genome
Exploration Research Project, Genomic JANET ESTEE KACHARMINA (), stnemtrapeD
of Pharmacology and Psychiatry, -revinU
secneicS Center (GSC), and Genome Sci-
sity of Pennsylvania Medical ,retneC Phila-
ence Laboratory, Riken Tsukuba Life Sci-
,aihpled Pennsylvania 40191
ence Center, ehT Institute of Physical and
Chemical Research (RIKEN), CREST, -aJ CHERYL EVANS (26), Genetics .etutitsnI Cam-
pan Science and Technology Corporation ,egdirb sttesuhcassaM 04120
(JST), Tsukuba, Ibaraki ,4700-503 Japan CARL FRIDDLE (29), Department of ,sciteneG
DEANNA M. CHURCH (6), Samuel Lunenfeld Yale ,ytisrevinU New Haven, Connecticut
hcraeseR Institute, Mount Sinai ,latipsoH 0256O
Toronto, Ontario M5G 1X5 adanaC KATHELEEN GARDINER (10), Eleanor Roose-
MATTHEW D. CLARK (13), Abt. Lehrach, Max velt Institute, Denver, Colorado 60208
Planck Institut far Molekulare Genetik, F. JOSEPH GERMINO (24), ytisrevinU of -ideM
Berlin (Dahlem) ,59141-D Germany lac Dentistry of New Jersey, Clinical Insti-
MAUREEN COLBERT (26), Genetics ,etutitsnI tute of New Jersey, New Brunswick, New
,egdirbmaC sttesuhcassaM 04120 yesreJ 10980
X CONTRIBUTORS TO VOLUME 303
TERAGRAM TEELF-NEDLOG (26), sciteneG In- HANS LEHRACH (13), Abt. Lehrach, Max
,etutits ,egdirbmaC sttesuhcassaM 04120 Planck Institut fiir Molekulare Genetik,
Berlin (Dahlem) ,59141-D Germany
EDIHIHSOY IKAZIHSAYAH (2), Laboratory for
Genome Exploration hcraeseR Project, -eG MIN LI (25), Departments of Physiology and
nomic secneicS Center (GSC), and Genome ,ecneicsorueN School of Medicine, Johns
Science Laboratory, Riken Tsukuba Life Hopkins ,ytisrevinU Baltimore, Maryland
ecneicS ,retneC The Institute of Physical and 50212
Chemical Research (RIKEN), CREST, -aJ GNEM LIU (3), Department of ,sciteneG Boyer
pan Science and Technology Corporation retneC for Molecular Medicine, Yale -inU
(JST), Tsukuba, Ibaraki ,4700-503 Japan versity School of Medicine, New Haven,
ANIRAHTAC HIORT (4), Department of -mehC tucitcennoC 01560
,yrtsi .W .M Keck Laboratory for Biomolec- LEAHCIM TTEVOL (8), The McDermott retneC
raM Imaging, New York ,ytisrevinU New for Human Growth and Development, -inU
York, New York 30001 ytisrev of Texas Southwestern Medical -neC
ret at ,sallaD Dallas, Texas 53257
UKUSAT HONJO (27), Department of lacideM
,yrtsimehC Faculty of ,enicideM Kyoto -inU YEGRES VONAYKUL (20), Shemyakin and
,ytisrev Sakyo-ku, Kyoto ,1058-606 Japan Ovchinnikov Institute of Bioorganic Chem-
,yrtsi Russian Academy of ,ecneicS V-437
LEAHCIM KNABUH (19), Trafford Centre for
Moscow ,178711 Russia
Medical Research, University of Sussex,
MIKAOJ GREBEDNUL (28), Department of -DiB
Brighton, Sussex BN1 9RY, England
yrtsimehc and Biotechnology, KTH, Royal
ENIREHTAC SGNIHCTUH (22), Trafford ertneC Institute of Technology, S-IO0 44 Stock-
for Medical Research, University of Sus- holm, Sweden
sex, Brighton, Sussex BN1 9RY, United
EDUALC-EIRAM REILOSRAM (23), Service ed
Kingdom
Biochimie et euqit~n~G ,erialuc~loM CEA/
IHSAKAT OTI (17), Human Genome ,retneC SACLAY, 91191 Gif Sur Yvette ,xedeC
Institute of Medical Science, University of ecnarF
Tokyo, Minato-ku, Tokyo ,9368-801 Japan
ENIREHTAK J. MARTIN (14), Dana-Farber
HTENNEK A. SBOCAJ (26), Genetics ,etutitsnI Cancer Institute, Boston, sttesuhcassaM
,egdirbmaC sttesuhcassaM 04120 31120
ARABRAB JUNG (18), Sidney Kimmel recnaC ESIO~NARF ~IDUAD-UEIHTAM (18, 21), Sidney
,retneC San Diego, California 12129 Kimmel recnaC ,retneC San Diego, -rofilaC
ain 12129
KERRY REHELLEK (26), Genetics Institute,
ENNYL V. MAYNE (22), Trafford Centre for
,egdirbmaC sttesuhcassaM 04120
Medical Research, University of ,xessuS
DLANOR KRIZ (26), Genetics Institute, Cam- Brighton, BNI 9RY, United Kingdom
,egdirb sttesuhcassaM 04120
LEAHCIM DNALLELCCM (18, 21), Sidney Kim-
SUNGAM NOSSRAL (28), Department of Bio- reel Cancer ,retneC naS Diego, ainrofilaC
yrtsimehc and Biotechnology, KTH, Royal 12129
Institute of Technology, S-IO0 44 Stock- JOHN M. McCoY (26), Genetics ,etutitsnI
holm, Sweden ,egdirbmaC sttesuhcassaM 04120
YuN-FA1 CHRIS LAU (20), Division of Cell DIVAD GREBREM (26), Astra hcraeseR ,retneC
and Developmental ,sciteneG Department Boston, Cambridge, Massachusetts -93120
of ,enicideM Vii Medical ,retneC ytisrevinU 9324
of ,ainrofilaC naS ,ocsicnarF San ,ocsicnarF
NEAL K. ZTlWOKSOM (24), Department of
ainrofilaC 12149
raluceloM sciteneG and ,ygoloiborciM -inU
DRAWDE R. LAVALLIE (26), Genetics Insti- ytisrev of Medical Dentistry of New ,yesreJ
,etut ,egdirbmaC sttesuhcassaM 04120 New Brunswick, New Jersey 10980
CONTRIBUTORS TO VOLUME 303 xi
RICHARD J. MURAL (5), Computational Biol- PETER SCnATZ (25), Affymax Research Insti-
ygo Section, Life Sciences Division, Oak tute, 400l Miranda Avenue. Palo Alto,
Ridge National Laboratory, Oak Ridge, CA 40349
eessenneT 13873
DAVID C. SCHWARTZ (4), Department of
TOMOYUKI NAKAMURA (27), Department of ,yrtsimehC .W .M Keck Laboratory for Bio-
Medical Chemistry, Faculty of ,enicideM molecular Imaging, New York ,v(isrevinU
Kyoto University, Sakyo-ku, Kyoto -606 New York, New York 30001
,1058 Japan
ANDR~ SENTENAC (23), Service ed Biochimie
PETER E. NEWBURGER (16), University of te euqit~n~G ,erialucdloM CEA/SACLA ,Y
sttesuhcassaM Medical Center, ,retsecroW 19119 Gi Sur Yvette ,xedeC ecnarF
sttesuhcassaM 50610
AMY SHEEnAN (29), Department of ,ygoloiB
JACOB ODEBERG (28), Department of Bio-
Yale ,ytisrevinU New Haven, Connecticut
yrtsimehc dna Bioteehnology, KTH, Royal
02560
Institute of Technology, S-IO0 44 Stock-
holm, Sweden PAUL D. SIEBERT (20), CLONTECH Labora-
,seirot Inc., Palo Alto, California -30349
GEORGIA D. PANOPOULOU (13), Abt. Leh-
0324
,hcar Max Planck Institut far Molekulare
Genetik, Berlin (Dahlem) D-14195, ANDREW D. SNOMMIS (8), ehT McDermott
ynamreG retneC for Human Growth dna Develop-
ARTHUR B. EEDRAP (14), Dana-Farber -naC ment, University of Texas Southwestern
rec Institute, Boston, sttesuhcassaM 31120 Medical Center ta Dallas, Dallas, Texas
53257
SATISH PARIMOO (9), Skin Biology hcraeseR
,retneC Johnson & Johnson, Skillman, New MICHAEL SNYDER (29), Department of Biol-
yesreJ 85580 ,ygo Yale ,ytisrevinU New Haven, -itcennoC
cut 02560
YATINDRA PRASHAR (15), Gene Logic, Inc.,
,grubsrehtiaG Maryland 87802 VIKKI GN~DLUAPS (26), Genetics ,etutitsnI
ANNE HANSEN REE (28), Department of -uT ,egdirbmaC "sttesuhcassaM 04120
mor Biology, Institute of Cancer ,hcraeseR STEFAN STAnL (28), Department of Biochem-
ehT Norwegian Radium Hospital, N-0310 yrtsi and Biotechnology, KTH, Royal -itsnI
,olsO Norway tute of Technology, S-IO0 44 Stockholm,
G. SHIRLEEN ROEDER (29), Department of Bi- nedewS
ology, Yale ,ytisrevinU New Haven, Con-
JEN STOVER (26), Genetics Institute, Cam-
necticut 02560
,egdirb sttesuhcassaM 04120
DVSTEIN ROSOK (28), Department of Immu-
NICOLE L. STmCKER (25), Department of Neu-
nology, Institute of Cancer Research, ehT
,ecneicsor School of ,enicideM Johns Hop-
Norwegian Radium Hospital, N-0310
kins ,ytisrevinU Baltimore, Maryland 50212
Oslo, Norway
PETRA ROsS-MACDONALD (29), Department Y. V. B. K. MAYNAMARBUS (3, 16), -trapeD
ment of ,sciteneG Boyer retneC for -uceloM
of Biology, Yale University, New Haven,
tucitcennoC 02560 ral Medicine, Yale University School of
,enicideM New Haven, Connecticut 63560
YOSHIYUKI SAKAK1 (17), Human Genome
and Gene Logic, Inc., ,grubsrehtiaG Mary-
,retneC Institute of lacideM ,ecneicS -revinU
land 87802
sity of Tokyo, Minato-ku, Tokyo -801
,9368 Japan KEI TASH1RO (27), Center for Molecular
Biology dna Genetics, Kyoto ,ytisrevinU
DAVID G. SCHATZ (19), Howard Hughes
Sakyo-ku, Kyoto ,7058-606 Japan
Medical Institute, Section of Immunobiol-
ogy, Yale University School of ,enicideM THOMAS TRENKLE (18, 21), Sidney Kimmel
New Haven, Connecticut 01560 recnaC ,retneC naS ,ogeiD ainrofilaC 12129
xii CONTRIBUTORS TO VOLUME 303
SAMOHT VOGT (18), University of Regens- NHOJ HSLEW (18, 21), Sidney Kimmel Cancer
burg, 93042 Regensburg, Germany Center, San Diego, California 12129
NAMREHS M. NAMSSIEW (9, ,51 16), Depart-
ment of Genetics, Boyer Center for Mo- KRAM J. NOSMAILLIW (26), MiUenium Phar-
lecular Medicine, Yale University School of maceuticals, Inc., Cambridge, Massachu-
Medicine, New Haven, Connecticut 63560 setts 93120
1 cDNA FROM SINGLE CELLS AND SUBCELLULAR REGIONS 3
1 Preparation of cDNA from Single Cells and
Subcellular Regions
yB TENAJ EETSE ,AN1MRAHCAK RETEP B. ,ONIRC and SEMAJ ENIWREBE
Introduction
The heterogeneity of cellular phenotypes in the brain has complicated
attempts to characterize alterations in gene expression under normal condi-
tions such as development and senescence, as well as in disease states.
Historically, the classification of cellular identity has been ascertained on
the basis of regional anatomic location (e.g., hippocampal CA1 neuron or
cerebellar Purkinje cell), individual morphology (e.g., pyramidal or stellate),
and electrophysiological properties as determined by whole-cell patch-
clamp techniques (e.g., excitatory or inhibitory). Neurons may also be
categorized on the basis of the proteins, neurotransmitters, or receptors
they express, as determined by immunohistochemistry or receptor autoradi-
ography. For example, neuronal progenitor cells express the embryonic
intermediate filament protein nestin, mature cells express neurofilament
proteins, and astroglial cells express glial fibrillary acidic protein and vi-
mentin.
In addition, the analysis of mRNA abundances in individual neurons
has been difficult because of the high level of cellular diversity in the brain.
That is, two cells that seem to be morphologically identical may express
different mRNAs at distinct abundances. Another complication in studying
neuronal gene expression in single ceils is that many important messages
are expressed in low abundance. Acquiring sufficient quantities of mRNA
to analyze gene expression has been difficult, as it si estimated that the
amount of mRNA in a single cell is between 1.0 and 0.1 pg. Among the
techniques used to study gene expression, in silu hybridization (ISH) can
render a view of mRNA localization in many cells simultaneously, both in
culture and in tissue sections. 1 However, one shortcoming of ISH is that
only one or two cRNA probes can be applied to a selected tissue section
at once, and thus, only one or two mRNAs can be assayed. Also, ISH
may not be sensitive enough to detect low-abundance mRNAs. n situ
transcription (IST), a technique that shares many features with ISH, in-
volves the synthesis of cDNA via the reverse transcription of polyade-
J. Eberwine, K. Valentino, and J. Barchas, "In Situ Hybridization in Neurobiology." Oxford
Press, New York, 1994.
Copyright © 1999 by Academic Press
All rights of reproduction in any form reserved.
METHODS NI ENZYMOLOGY, VOL. 303 99/9786~6700 $30.00
4 cDNA PREPARATION 1
nylated poly(A) + mRNA in fixed tissues or live cells in culture. 5-2 There
are two technologies that begin by an initial cDNA synthesis step and can
be used to amplify mRNA from single cells, namely, reverse transcriptase-
polymerase chain reaction (RT-PCR) 8-6 and antisense RNA (aRNA) am-
plificationY RT-PCR uses Taq DNA polymerase and is therefore an expo-
nential amplification procedure, making accurate quantification of mRNA
abundances problematic. In contrast, the aRNA amplification technique
amplifies mRNA in a linear fashion, using T7 RNA polymerase, and permits
direct quantification of the relative abundances of individual mRNA spe-
cies. The ability to quantitate mRNA levels is critical, because expression
analysis of multiple genes simultaneously in single cells provides an impor-
tant avenue toward understanding neuronal functioning and the molecular
pathophysiology of specific diseases. Indeed, because many mRNAs are
expressed in only a few copies in single cells, and there is a high level of
cellular diversity, it is imperative that studies of neuronal gene expression
be accomplished at the single-cell level.
Overview of cDNA Synthesis and Antisense RNA Amplification
The analysis of mRNA populations within single cells became possible
with the advent of the amplified, antisense (aRNA) amplification technique.
The mRNA population in a single cell can be amplified such that its size
and complexity are proportionally represented in the resultant amplified
aRNA population. 4 A schematic diagram of the aRNA amplification
method is shown in Fig. .1 The first step in the synthesis of cDNA is
hybridization of an oligo(dT) primer, oligo(dT)z4-T7 to the endogenous
poly(A) + mRNA. The oligo(dT)e4-T7 contains the bacteriophage T7 RNA
polymerase promoter sequence 5' to the polythymidine segment. In addi-
tion, individual mRNAs can be amplified by using primers complementary
to specific mRNAs of interest, and that have an attached T7 RNA polymer-
ase promoter sequence, to prime cDNA synthesis. Avian myeloblastosis
reverse transcriptase (AMV-RT; Seikagaku America, Ijamsville, MD) binds
2 .L Tecott, .J ,sahcraB dna .J ,eniwrebE Science ,042 1661 .)8891(
~" .I Zangger, .L Tecott, dna .J ,eniwrebE Technique ,1 801 .)9891(
4 .R VanGelder, .M ,wortsaZnov .A Yool, .W Dement, .J ,sahcraB dna .J ,eniwrebE Proc.
Natl. Acad. Sci. U.S.A. ,78 3661 .)0991(
5 j. ,eniwrebE .H Yeh, .K ,orihsayiM .Y Cao, .S Nair, .R Finnell, .M Zettel, dna .P ,nameloC
Proc. Natl. Acad. Sci. U.S.A. ,98 0103 .)2991(
6 j. ,regniS-maS .M Robinson, .A Bellvue, .M ,nomiS dna .A ,sggiR Nucleic Acids Res. ,81
5521 .)0991(
7 j. Robinson and .M ,nomiS Nucleic Acids Res. ,91 7551 .)1991(
s .C ,kerazcwO .P ,sirraH-zeuqirnE dna .N Proudfoot, Nucleic Acids Res. ,02 158 .)2991(
mRNA 5' AAA-3'
I sPTNd Reverse Transcription of ANRm
7T-42Td-ogilo
TR-VMA
mRNA 5' I I,,I I I AAA-3'
'95 ° 3 setunim Denaturation of ANDc-ANRm Hybrid
t st strand cDNA 3' i .... TTT-'r7-5'
I sPTNd dn2 Strand Synthesis
wonelK
4T AND esaremyloP
22°2:,?. i
Sl esaelcuN $t Nuelease Treatment
~ sPTNdK wone Blunt-ending
2nd strand cDNA 5' ,,, ( -dAdAdA-T7 3'
ts1 strand eDNA 3' 1 I, I I, I ~TTT-I'7-5'
I sPTN ts1 round eRNA Amplification
7T ANR esaremyloP 103-told
aRNA3' - ,-. ____IIUU-5'
aRNA 3' _ ,,, UUU-5'
aRNA 3' ~ UU-5'
sPTNd Reverse Transcription of ANRa
~ TI R-VM modnAaR cificebS/sremaxeH ANDc sremirP
~95 ,o 3 setunim noitarutaneD fo ANDcIANRa Hybrid
ts1 strand cDNA 5' dAdAdA-3'
I sPTNd dn2 Strand Synthesis
7T-42Td-Ogilo
wonelK
r 4T AND esaremyloP
I,i:ANNDDcC~n2artrstsdt121 I I I I,. I I I 7T517:ATd.~(I "3'
I sPTN dn2 Round aRNA Amplification
7T ANR Polymerase 103X103=I06 Fold
aRNA 3' UUU-5'
aRNA 3' I IUU-5'
aRNA 3' ~,. J I IUU-5'
ANRa eborP
,RCP RCP-TR
ANDc yrarbiL
laitnerefliD yalpsiD
F(~. .1 Schematic of first and second rounds of antisense RNA amplification proced
6 cDNA NOITARAPERP 1
to the primer/template oligo(dT)24-T7/mRNA complex and copies single-
stranded mRNA into cDNA, such that each cDNA contains a T7 RNA
polymerase promoter site. This results in an mRNA/cDNA duplex that is
then denatured. The cDNA is made double stranded by hairpin loop sec-
ond-strand synthesis using Klenow fragment of Escherichia coli DNA poly-
merase I and T4 DNA polymerase. The hairpin loop of the double-stranded
cDNA is cleaved with $1 nuclease and blunt ended with Klenow fragment.
The T7 RNA polymerase promoter region, once double stranded, is func-
tional, at which point the cDNA can serve as a template for aRNA synthesis
with the addition of T7 RNA polymerase. To yield higher levels of aRNA,
a second round of amplification is performed in which the aRNA serves
as a template for cDNA synthesis, and is reverse transcribed with AMV-
RT, resulting in an aRNA-cDNA hybrid. After heat denaturation, the
cDNA is made double stranded, drop dialyzed, and reamplified using T7
RNA polymerase, as described above.
By incorporating a radiolabel, the second-round aRNA product can be
used as a probe for reverse Northern blots to create mRNA expression
profiles of single cells. The aRNA can also be used as a template for RT-
PCR or cloned to construct a single-cell cDNA library. In addition, single-
or double-stranded cDNA processed through one round of the aRNA
procedure can serve as a template for differential display of single cells to
identify novel, differentially expressed genes. 9 The aRNA amplification
technique has been used to synthesize aRNA from mRNA in single dissoci-
ated live neurons in culture, subcellular regions such as dendrites °1 and
growth cones, 11 single cells from fixed tissue, 21 and single cells from live
slice preparations. 41"31 When using primary cultures of neurons the complex
synaptic interactions required for proper CNS functioning are disrupted.
The use of live slice preparations facilitates the study of neuronal gene
expression in a system that preserves many synaptic and glial connections.
Furthermore, whole-cell electrophysiological recordings can be performed
in conjunction with the aRNA amplification procedure in single cells from
dissociated cultures 6351'5'4 or live slice preparations, 41'3a Electrophysiologi-
9 p. Liang and A. Pardee, Science 257, 967 (1992).
01 K. Miyashiro, M. Dichter, and J. Eberwine, Proc. Natl. Acad. Sci. U.S.A. 91, 10800 (1994).
11 p. Crino and J. Eberwine, Neuron 17, 1173 (1996).
21 p. Crino, M. Dichter, J. Trojanowski, and J. Eberwine, Proc. Natl. Acad. Sci. U.S.A. 93,
14152 (1996).
3a .S Mackler, B. Brooks, and J. Eberwine, Neuron ~9 539 (1992).
41 S. Mackler and J. Eberwine, Mol. Pharmacol. 44, 308 (1993).
51 j. Surmeier, J. Eberwine, C. Wilson, Y. Cao, A. Stefani, and .S Kitai, Proc. Natl. Acad. Sci.
U.S.A. 89, 10178 (1992).
61 j. Bargas, A. Howe, J. Eberwine, Y. Cao, and D. Surmeier, J. Neurosci. 14, 6667 (1994).
1 cDNA 7
FROM SINGLE CELLS AND SUBCELLULAR REGIONS
cal data can then be correlated with coordinate changes in gene expression
in single, morphologically identified cells.
Preparation of cDNA and Antisense RNA Amplification of mRNA from
Single Dissociated Cells in Culture
In these studies a whole-cell patch electrode is utilized to harvest the
mRNA from a single dissociated hippocampal neuron (embryonic day
20-21). 71 It is imperative that all reagents and the working environment
be kept RNase free to prevent RNA degradation throughout the aRNA
amplification procedure. Water should be treated with diethylpyrocarbon-
ate (DEPC)J 8 The microelectrode is backfilled with a solution containing
the reagents necessary for first-strand cDNA synthesis in the following final
concentrations: a 250/xM concentration each of deoxyadenosine triphos-
phate (dATP), deoxyguanosine triphosphate (dGTP), thymidine triphos-
phate (TTP), and deoxycytosine triphosphate (dCTP); 5 ng//xl oligo(dT)24-
T7 (100 ng//xl) primer containing a T7 RNA polymerase promoter region
AAACGACGGCCAGTGAATTGTAATACGACTCACTA-
TAGGCGC(T)24-T7; and 0.5 U//xl AMV-RT (20 U//xl) in 1.0× electrode
buffer (120 mM KC1, 1 mM MgC12, and 10 mM HEPES, pH 7.3). Whole-
cell recordings may be done at this point if desired; however, modification
of the electrode buffer may be necessary. The contents of a single, cultured,
hippocampal cell are then gently aspirated into the microelectrode (Fig.
2). The entire contents of the patch pipette are then expelled into a micro-
centrifuge tube containing a 250/xM concentration each of dATP, dGTP,
TTP, and dCTP; 5 mM dithiothreitol (DTT); 1 U//xl RNasin; and 0.5
U//xl AMV-RT in the above-mentioned buffer, adjusted to pH 8.3 to opti-
mize for AMV-RT activity. First-strand cDNA synthesis is accomplished
by incubating the preceding reagents with cellular contents for 60-90 min
at temperatures between 37 and 50 ° .
Alternatively, oligo(dT)24-T7 primers coupled to magnetic porous glass
beads (CPG, Lincoln Park, N J) that are attached via a primary amine
linkage can be utilized. 91 The use of the magnetic beads obviates the need
for phenol-chloroform extraction and ethanol precipitation. If the magnetic
beads attached to the oligo(dT)24-T7 are not used, the sample is phenol-
chloroform extracted by adding 0.5 vol of TE 10 mM Tris-HC1 (pH 8.0)
and 1 mM EDTA-saturated phenol, 0.5 vol of chloroform, and 1.0 vol of
71 j. retlahcuB dna .M ,rethciD Brain Res. Bull ,62 333 .)1991(
s~ .J ,koorbmaS .E ,hcstirF dna .T ,sitainaM raluceloM" :gninolC A Laboratory ".launaM
dloC gnirpS Harbor ,sserP Cold gnirpS Harbor, weN York, .9891
91 j. ,eniwrebE Biotechniques ,02 485 .)6991(
