cdna preparation and characterization

Preface 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 Chemistry, W. M. Keck Laboratory for Bio- molecular Imaging, New York University, New York, New York 10003 NAMADEV BASKARAN (3, 16), Genome Thera- peutics Corporation, Waltham, Massachu- setts 02453 ALASTAIR J. H. BROWN (22), Trafford Centre for Medical Research, University of Sussex, Brighton, BN1 9RY, United Kingdom PATRICK O. BROWN (12), Department of Bio- chemistry, Stanford University School of Medicine and Howard Hughes Medical In- stitute, Stanford, California 94305-5428 ALAN J. BUCKLER (6), Axys Pharmaceuticals, La Jolla, California 92037 JULIAN F. BURKE (22), School of Biological Sciences, University of Sussex, Brighton, BN1 9RY, United Kingdom KONRAD B(3SSOW (13), Abt. Lehrach, Max Planck Institut far Molekulare Genetik, Berlin (Dahlem) D-14195, Germany DOLORES J. CAHILL (13), Abt. Lehrach, Max Planck Institut far Molekulare Genetik, Berlin (Dahlem) D-14195, Germany PIERO CARNINCI (2), Laboratory for Genome Exploration Research Project, Genomic Sciences Center (GSC), and Genome Sci- ence Laboratory, Riken Tsukuba Life Sci- ence Center, The Institute of Physical and Chemical Research (RIKEN), CREST, Ja- pan Science and Technology Corporation (JST), Tsukuba, Ibaraki 305-0074, Japan DEANNA M. CHURCH (6), Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5 Canada MATTHEW D. CLARK (13), Abt. Lehrach, Max Planck Institut far Molekulare Genetik, Berlin (Dahlem) D-14195, Germany MAUREEN COLBERT (26), Genetics Institute, Cambridge, Massachusetts 02140 LISA A. COLLINS-RAcIE (26), Genetics Insti- tute, Cambridge, Massachusetts 02140 PETER B. CRINO (1), Department of Neurol- ogy, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104 JOHAN T. DEN DUNNEN (7), Department of Human Genetics, Leiden University Medi- cal Center, 2333 Al Leiden, The Netherlands LUDA DIATCHENKO (20), CLONTECH Laboratories, Inc., Palo Alto, California 94303-4230 RADOJE DRMANAC (11), Hyseq, Inc., Sunny- vale, California 94086 SNEZANA DRMANAC (11), Hyseq, Inc., Sunny- vale, California 94086 McKEOUGH DUCKETT (26), Genetics Institute, Cambridge, Massachusetts 02140 JAMES EBERWINE (1), Departments of Phar- macology and Psychiatry, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104 MICHAEL B. EISEN (12), Departments of Ge- netics and Biochemistry, Stanford Univer- sity School of Medicine, Stanford, Califor- nia 94305-5428 JANET ESTEE KACHARMINA (]), Departments of Pharmacology and Psychiatry, Univer- sity of Pennsylvania Medical Center, Phila- delphia, Pennsylvania 19104 CHERYL EVANS (26), Genetics Institute. Cam- bridge, Massachusetts 02140 CARL FRIDDLE (29), Department of Genetics, Yale University, New Haven, Connecticut O6520 KATHELEEN GARDINER (10), Eleanor Roose- velt Institute, Denver, Colorado 80206 F. JOSEPH GERMINO (24), University of Medi- cal Dentistry of New Jersey, Clinical Insti- tute of New Jersey, New Brunswick, New Jersey 08901 X CONTRIBUTORS TO VOLUME 303 MARGARET GOLDEN-FLEET (26), Genetics In- stitute, Cambridge, Massachusetts 02140 YOSHIHIDE HAYASHIZAKI (2), Laboratory for Genome Exploration Research Project, Ge- nomic Sciences Center (GSC), and Genome Science Laboratory, Riken Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), CREST, Ja- pan Science and Technology Corporation (JST), Tsukuba, Ibaraki 305-0074, Japan CATHARINA HIORT (4), Department of Chem- istry, W. M. Keck Laboratory for Biomolec- Mar Imaging, New York University, New York, New York 10003 TASUKU HONJO (27), Department of Medical Chemistry, Faculty of Medicine, Kyoto Uni- versity, Sakyo-ku, Kyoto 606-8501, Japan MICHAEL HUBANK (19), Trafford Centre for Medical Research, University of Sussex, Brighton, Sussex BN1 9RY, England CATHERINE HUTCHINGS (22), Trafford Centre for Medical Research, University of Sus- sex, Brighton, Sussex BN1 9RY, United Kingdom TAKASHI ITO (17), Human Genome Center, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan KENNETH A. JACOBS (26), Genetics Institute, Cambridge, Massachusetts 02140 BARBARA JUNG (18), Sidney Kimmel Cancer Center, San Diego, California 92121 KERRY KELLEHER (26), Genetics Institute, Cambridge, Massachusetts 02140 RONALD KRIZ (26), Genetics Institute, Cam- bridge, Massachusetts 02140 MAGNUS LARSSON (28), Department of Bio- chemistry and Biotechnology, KTH, Royal Institute of Technology, S-IO0 44 Stock- holm, Sweden YuN-FA1 CHRIS LAU (20), Division of Cell and Developmental Genetics, Department of Medicine, Vii Medical Center, University of California, San Francisco, San Francisco, California 94121 EDWARD R. LAVALLIE (26), Genetics Insti- tute, Cambridge, Massachusetts 02140 HANS LEHRACH (13), Abt. Lehrach, Max Planck Institut fiir Molekulare Genetik, Berlin (Dahlem) D-14195, Germany MIN LI (25), Departments of Physiology and Neuroscience, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205 MENG LIU (3), Department of Genetics, Boyer Center for Molecular Medicine, Yale Uni- versity School of Medicine, New Haven, Connecticut 06510 MICHAEL LOVETT (8), The McDermott Center for Human Growth and Development, Uni- versity of Texas Southwestern Medical Cen- ter at Dallas, Dallas, Texas 75235 SERGEY LUKYANOV (20), Shemyakin and Ovchinnikov Institute of Bioorganic Chem- istry, Russian Academy of Science, V-437 Moscow 117871, Russia JOAKIM LUNDEBERG (28), Department of BiD- chemistry and Biotechnology, KTH, Royal Institute of Technology, S-IO0 44 Stock- holm, Sweden MARIE-CLAUDE MARSOLIER (23), Service de Biochimie et G~n~tique Mol~culaire, CEA/ SACLAY, 91191 Gif Sur Yvette Cedex, France KATHERINE J. MARTIN (14), Dana-Farber Cancer Institute, Boston, Massachusetts 02113 FRAN~OISE MATHIEU-DAUDI~ (18, 21), Sidney Kimmel Cancer Center, San Diego, Califor- nia 92121 LYNNE V. MAYNE (22), Trafford Centre for Medical Research, University of Sussex, Brighton, BNI 9RY, United Kingdom MICHAEL MCCLELLAND (18, 21), Sidney Kim- reel Cancer Center, San Diego, California 92121 JOHN M. McCoY (26), Genetics Institute, Cambridge, Massachusetts 02140 DAVID MERBERG (26), Astra Research Center, Boston, Cambridge, Massachusetts 02139- 4239 NEAL K. MOSKOWlTZ (24), Department of Molecular Genetics and Microbiology, Uni- versity of Medical Dentistry of New Jersey, New Brunswick, New Jersey 08901 CONTRIBUTORS TO VOLUME 303 xi RICHARD J. MURAL (5), Computational Biol- ogy Section, Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 TOMOYUKI NAKAMURA (27), Department of Medical Chemistry, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606- 8501, Japan PETER E. NEWBURGER (16), University of Massachusetts Medical Center, Worcester, Massachusetts 01605 JACOB ODEBERG (28), Department of Bio- chemistry and Bioteehnology, KTH, Royal Institute of Technology, S-IO0 44 Stock- holm, Sweden GEORGIA D. PANOPOULOU (13), Abt. Leh- rach, Max Planck Institut far Molekulare Genetik, Berlin (Dahlem) D-14195, Germany ARTHUR B. PARDEE (14), Dana-Farber Can- cer Institute, Boston, Massachusetts 02113 SATISH PARIMOO (9), Skin Biology Research Center, Johnson & Johnson, Skillman, New Jersey 08558 YATINDRA PRASHAR (15), Gene Logic, Inc., Gaithersburg, Maryland 20878 ANNE HANSEN REE (28), Department of Tu- mor Biology, Institute of Cancer Research, The Norwegian Radium Hospital, N-0310 Oslo, Norway G. SHIRLEEN ROEDER (29), Department of Bi- ology, Yale University, New Haven, Con- necticut 06520 DVSTEIN ROSOK (28), Department of Immu- nology, Institute of Cancer Research, The Norwegian Radium Hospital, N-0310 Oslo, Norway PETRA ROsS-MACDONALD (29), Department of Biology, Yale University, New Haven, Connecticut 06520 YOSHIYUKI SAKAK1 (17), Human Genome Center, Institute of Medical Science, Univer- sity of Tokyo, Minato-ku, Tokyo 108- 8639, Japan DAVID G. SCHATZ (19), Howard Hughes Medical Institute, Section of Immunobiol- ogy, Yale University School of Medicine, New Haven, Connecticut 06510 PETER SCnATZ (25), Affymax Research Insti- tute, 400l Miranda Avenue. Palo Alto, CA 94304 DAVID C. SCHWARTZ (4), Department of Chemistry, W. M. Keck Laboratory for Bio- molecular Imaging, New York Universi(v, New York, New York 10003 ANDR~ SENTENAC (23), Service de Biochimie et G~n~tique Moldculaire, CEA/SACLA Y, 91191 Gi[ Sur Yvette Cedex, France AMY SHEEnAN (29), Department of Biology, Yale University, New Haven, Connecticut 06520 PAUL D. SIEBERT (20), CLONTECH Labora- tories, Inc., Palo Alto, California 94303- 4230 ANDREW D. SIMMONS (8), The McDermott Center for Human Growth and Develop- ment, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235 MICHAEL SNYDER (29), Department of Biol- ogy, Yale University, New Haven, Connecti- cut 06520 VIKKI SPAULD~NG (26), Genetics Institute, Cambridge, Massachusetts" 02140 STEFAN STAnL (28), Department of Biochem- istry and Biotechnology, KTH, Royal Insti- tute of Technology, S-IO0 44 Stockholm, Sweden JEN STOVER (26), Genetics Institute, Cam- bridge, Massachusetts 02140 NICOLE L. STmCKER (25), Department of Neu- roscience, School of Medicine, Johns Hop- kins University, Baltimore, Maryland 21205 Y. V. B. K. SUBRAMANYAM (3, 16), Depart- ment of Genetics, Boyer Center for Molecu- lar Medicine, Yale University School of Medicine, New Haven, Connecticut 06536 and Gene Logic, Inc., Gaithersburg, Mary- land 20878 KEI TASH1RO (27), Center for Molecular Biology and Genetics, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan THOMAS TRENKLE (18, 21), Sidney Kimmel Cancer Center, San Diego, California 92121 xii CONTRIBUTORS TO VOLUME 303 THOMAS VOGT (18), University of Regens- burg, 93042 Regensburg, Germany SHERMAN M. WEISSMAN (9, 15, 16), Depart- ment of Genetics, Boyer Center for Mo- lecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06536 JOHN WELSH (18, 21), Sidney Kimmel Cancer Center, San Diego, California 92121 MARK J. WILLIAMSON (26), MiUenium Phar- maceuticals, Inc., Cambridge, Massachu- setts 02139 [ 1] cDNA FROM SINGLE CELLS AND SUBCELLULAR REGIONS 3 [1] Preparation of cDNA from Single Cells and Subcellular Regions By JANET ESTEE KACHARM1NA, PETER B. CRINO, and JAMES EBERWINE 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 is estimated that the amount of mRNA in a single cell is between 0.1 and 1.0 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 IN ENZYMOLOGY, VOL. 303 0076~6879/99 $30.00 4 cDNA PREPARATION [ 1] nylated [poly(A) +] mRNA in fixed tissues or live cells in culture. 2-5 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) 6-8 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. Barchas, and J. Eberwine, Science 240, 1661 (1988). "~ I. Zangger, L. Tecott, and J. Eberwine, Technique 1, 108 (1989). 4 R. VanGelder, M. vonZastrow, A. Yool, W. Dement, J. Barchas, and J. Eberwine, Proc. Natl. Acad. Sci. U.S.A. 87, 1663 (1990). 5 j. Eberwine, H. Yeh, K. Miyashiro, Y. Cao, S. Nair, R. Finnell, M. Zettel, and P. Coleman, Proc. Natl. Acad. Sci. U.S.A. 89, 3010 (1992). 6 j. Sam-Singer, M. Robinson, A. Bellvue, M. Simon, and A. Riggs, Nucleic Acids Res. 18, 1255 (1990). 7 j. Robinson and M. Simon, Nucleic Acids Res. 19, 1557 (1991). s C. Owczarek, P. Enriquez-Harris, and N. Proudfoot, Nucleic Acids Res. 20, 851 (1992). mRNA 5' mRNA 5' t st strand cDNA 3' AAA-3' I dNTPs oligo-dT24-T7 AMV-RT AAA-3' I I,,I I I '95 ° 3 minutes i TTT-'r7-5' I dNTPs Klenow T4 DNA Polymerase Reverse Transcription of mRNA Denaturation of mRNA-cDNA Hybrid 2nd Strand Synthesis 22°2:,?. i Sl Nuclease $t Nuelease Treatment dNTPs Blunt-ending ~ K enow 2nd strand cDNA 5' ,,, ( -dAdAdA-T7 3' 1st strand eDNA 3' 1 I, I I I, [ ~TTT-I'7-5' I NTPs 1st round eRNA Amplification T7 RNA Polymerase 103-told aRNA3' - , ____IIUU-5' aRNA 3' _ ,,, UUU-5' aRNA 3' ~ UU-5' dNTPs Reverse Transcription of aRNA I Random Hexamers/Sbecific cDNA Primers ~ AMV-RT ~95 o, Denaturation of aRNAIcDNA Hybrid 3 minutes 1st strand cDNA 5' dAdAdA-3' I dNTPs 2nd Strand Synthesis oligO-dT24-T7 Klenow r T4 DNA Polymerase 121tdstsrtra2n~CcDDNNA:i,I I I I .,I I I I I(~.dTA:715T7 "3' I NTPs 2nd Round aRNA Amplification T7 RNA Polymerase 103X103=I06 Fold aRNA 3' UUU-5' aRNA 3' I IUU-5' aRNA 3' .,~ J I IUU-5' aRNA Probe PCR, RT-PCR cDNA Library Dilferential Display F[(~. 1. Schematic of first and second rounds of antisense RNA amplification proced 6 cDNA PREPARATION [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, 12 and single cells from live slice preparations. 13"14 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 4'5'1536 or live slice preparations, a3'14 Electrophysiologi- 9 p. Liang and A. Pardee, Science 257, 967 (1992). 10 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). 12 p. Crino, M. Dichter, J. Trojanowski, and J. Eberwine, Proc. Natl. Acad. Sci. U.S.A. 93, 14152 (1996). a3 S. Mackler, B. Brooks, and J. Eberwine, Neuron 9~ 539 (1992). 14 S. Mackler and J. Eberwine, Mol. Pharmacol. 44, 308 (1993). 15 j. Surmeier, J. Eberwine, C. Wilson, Y. Cao, A. Stefani, and S. Kitai, Proc. Natl. Acad. Sci. U.S.A. 89, 10178 (1992). 16 j. Bargas, A. Howe, J. Eberwine, Y. Cao, and D. Surmeier, J. Neurosci. 14, 6667 (1994). [ 1] cDNA FROM SINGLE CELLS AND SUBCELLULAR REGIONS 7 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). 17 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. 19 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 0.1 vol of 17 j. Buchalter and M. Dichter, Brain Res. Bull 26, 333 (1991). ~s J. Sambrook, E. Fritsch, and T. Maniatis, "Molecular Cloning: A Laboratory Manual." Cold Spring Harbor Press, Cold Spring Harbor, New York, 1989. 19 j. Eberwine, Biotechniques 20, 584 (1996). [...]... mRNA and position of the two diol groups at the 5' end(cap) and the 3' end of the mRNA (C) Strategy for the preparation of the cDNA library FL, Full-length first-strand cDNA: NFL, non-full-length cDNA 22 cDNA PREPARATION [2] it possible to synthesize the second strand with high efficiency even in the case of long cDNAs Because this protocol employs 5-methyl-dCTP instead of dCTP for the first-strand cDNA. .. distinguish between real and artifactual bands corresponding to mRNAs amplified by differential display To generate gene expression profiles of mRNA populations within restricted subcellular regions, labeled aRNA can be used to probe reverse Northern blots containing a variety of candidate cDNAs, or cDNA arrays containing from just a few to several thousand candidate cDNAs 3°31 cDNA Synthesis and Antisense RNA... pelleted, air dried, and resuspended in 20/xl of DEPC-treated water The first-strand mRNA -cDNA hybrid is heat denatured at 95° for 3 min and placed on ice In this protocol, second-strand synthesis is accomplished by allowing the cDNA to serve as its own primer and to hairpin at its 3' end 2° However, alternative procedures such as that of Gubler and Hoffman,2~ or 3'-end tailing and specific priming,22... Sigma[; DNase- and RNase-free glycogen (Boehringer Mannheim, Indianapolis, IN); transfer RNA (tRNA, E coli; Sigma) To ensure the absence of DNA, RNase-free DNase I (Promega) digestion is done, followed by SDS-proteinase K treatment, phenol-chloroform and chloroform extraction, and ethanol precipitation following standard procedures; first- and second-strand cDNA primers (see text for sequence and preparation) ... Vector Preparation Before starting the preparation of a cDNA library, prepare and test a high-efficiency, low-background cloning vector As the cDNA fraction is radiolabeled, it becomes unstable and undergoes degradation if cloning is delayed This will more dramatically affect the cloning of the longer than the shorter clones, resulting in a size bias and overrepresentation of shorter clones 24 cDNA PREPARATION. .. ZapII (Stratagene)? °'n Enzymes and buffers: Restriction enzymes and reaction buffers, SstI (GIBCO-BRL, Gaithersburg, MD) and X h o I [Takara (Ohtsu, Japan) or New England BioLabs (Beverly, MA)];/3-agarase and reac7 p S Nelson, T S Papas, and C W Schweinfest, Nucleic Acids Res 3, 681 (1993) 8 A Kitamura and P Carninci, unpublished observation (1997) 9 j Sambrook, E F Fritsch, and T Maniatis, "Molecular... results in longer cDNAs, higher representation of long, full-length cDNAs in the library, and an overall higher yield of the recovered full-length cDNA The other problem in library preparation is that there have not been effective methods for selection of full-length cDNAs from incompletely extended cDNAs To solve this, we introduced a modified "biotinylated cap trapper" to select full-length cDNAs after... synthesize double-stranded cDNA For self-primed second-strand synthesis, the following reagents are added to the denatured cDNA in a final reaction volume of 40/xl: a 250 /zM concentration each of dATP, dGTP, TTP, and dCTP; 1 U of T4 DNA polymerase; and 2 U of Klenow fragment in 1.0× second-strand buffer [100 mM Tris-HC1 (pH 7.4), 20 mM KC1, 10 mM MgCI2,40 mM ( N H 4 ) 2 8 0 4 , and 5 mM DTT] The reaction... ready to prime the first-strand cDNA synthesis cDNA Preparation: First-Strand Synthesis The average size of the first-strand cDNA when the RT is thermostabilized by addition of trehalose is greater than that of cDNA synthesized under standard conditions The highest performance of the thermostabilized RT is obtained with temperature cycling between 55 and 60°.2 Before starting the reaction, a thermal... mRNAs, and the other on the terminal 3' end of any R N A (Fig 1B) The mRNA is biotinylated after the first-strand cDNA synthesis 5 In the case of incomplete first-strand synthesis, there is a single-stranded RNA (ssRNA) that connects the biotinylated cap and the truncated c D N A mRNA hybrid (Fig 1C) This ssRNA can be digested by RNase I, an ssRNA-specific ribonuclease, in the case of truncated cDNAs, . Northern blots containing a variety of candidate cDNAs, or cDNA arrays containing from just a few to several thousand candidate cDNAs. 3°31 cDNA Synthesis and Antisense RNA Amplification of mRNA. Transcription of aRNA I Random Hexamers/Sbecific cDNA Primers ~ AMV-RT ~95 o, Denaturation of aRNAIcDNA Hybrid 3 minutes 1st strand cDNA 5' dAdAdA-3' I dNTPs 2nd Strand Synthesis oligO-dT24-T7. 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