RNA Amplification in Brain Tissues

1 Center for Dementia Research, Nathan Kline Institute, New YorkUniversity School of Medicine, Orangeburg, NY 10962.

2 Department of Psychiatry & Neuroscience, New York UniversitySchool of Medicine, Orangeburg, NY 10962.

3 Address reprint request to: Stephen D. Ginsberg, Ph.D., Centerfor Dementia Research, Nathan Kline Institute, New York Uni-versity School of Medicine, 140 Old Orangeburg Road, Orange-burg, NY 10962. Tel: (845)398-2170; Fax: (845)398-5422; E-mail:[email protected]

RNA Amplification in Brain Tissues

Stephen D. Ginsberg1,2,3 and Shaoli Che1

(Accepted August 1, 2002)

Recent developments in gene array technologies, specifically cDNA microarray platforms, havemade it easier to try to understand the constellation of gene alterations that occur within theCNS. Unlike an organ that is comprised of one principal cell type, the brain contains a multi-plicity of both neuronal (e.g., pyramidal neurons, interneurons, and others) and noneuronal (e.g.,astrocytes, microglia, oligodendrocytes, and others) populations of cells. An emerging goal ofmodern molecular neuroscience is to sample gene expression from similar cell types within adefined region without potential contamination by expression profiles of adjacent neuronal sub-types and noneuronal cells. At present, an optimal methodology to assess gene expression is toevaluate single cells, either identified physiologically in living preparations, or by immunocyto-chemical or histochemical procedures in fixed cells in vitro or in vivo. Unfortunately, the quan-tity of RNA harvested from a single cell is not sufficient for standard RNA extraction methods.Therefore, exponential polymerase-chain reaction (PCR) based analyses and linear RNA ampli-fications, including a newly developed terminal continuation (TC) RNA amplification method-ology, have been used in combination with single cell microdissection procedures to enable theuse of cDNA microarray analysis within individual populations of cells obtained from post-mortem brain samples as well as the brains of animal models of neurodegeneration.

KEY WORDS: cDNA microarray; single cell analysis; expression profiling; RNA amplification.

Neurochemical Research, Vol. 27, No. 10, October 2002 (© 2002), pp. 981–992

9810364-3190/02/1000–0981/0 © 2002 Plenum Publishing Corporation

ideal structure to evaluate relationships between func-tional genomics and expression data. Contemporarymolecular-based techniques have enabled the study ofboth genomic DNA and RNA populations from fresh,fixed, and paraffin-embedded tissues (1–3). For exam-ple, upon identification of novel disease-related genes,retrospective mutation analysis in genomic DNA sam-ples from archived tissues is tenable. Moreover, expres-sion profiling of mRNA provides a real-time evaluationof gene expression. One mitigating factor is that thebrain is a complex structure with heterogeneous neu-ronal and noneuronal cellular populations. Each dis-tinct cell type is likely to have a signature “molecularfingerprint” under normal and neuropathological con-ditions. Thus, the pattern of mRNA expression insingle cells or a population of cells may be more in-formative than regional expression patterns. Specifi-cally, the molecular underpinnings that determinewhether a given neuronal population is vulnerable to

INTRODUCTION

Advances in molecular genetics have led to se-quencing of the human genome, and whole genomesequencing for other species is ongoing. Moreover, ex-pression data is becoming available for many diversetissues throughout the body, allowing for exciting hypo-thesis testing of critical concepts such as development,differentiation, homeostasis, and ultimately, diseasepathogenesis. The central nervous system (CNS) is an

neurodegeneration, i.e., “selective vulnerability”, maybe elucidated by single cell mRNA analyses (4,5).

Gene Expression Profiling Using Fixed and FrozenTissues

Evaluation of postmortem and/or perfused fixedtissues is highly desirable due to the large quantitiesof human and animal brain tissues that are archivedwithin individual laboratories and in brain banks (1,6).Currently, there is no specific ‘consensus’ protocol foroptimal tissue fixation for molecular-based studies.Several laboratories have reported significant successmining cDNA array data using tissue samples fromhuman brains fixed in both crosslinking fixatives suchas paraformaldehyde and 10% neutral-buffered forma-lin as well as precipitating fixatives such as 70% eth-anol buffered with 150 mM sodium chloride (7–12).Additional variables, including antemortem charac-teristics, duration of fixation, and length of storageshould be taken into account prior to initiating expres-sion profiling studies (1). One method useful for eval-uating the presence and quality of RNA species intissue sections is acridine orange (AO) histofluores-cence. AO is a fluorescent dye that intercalates selec-tively into nucleic acids (13,14), and has been used todetect RNA and DNA in brain tissues (15,16). Uponexcitation with ultraviolet spectra, AO that intercalatesinto RNA emits an orange-red fluorescence, whereasAO that intercalates into DNA emits a yellowish-greenfluorescence. In brain tissue sections, AO histofluo-rescence detects the presence of RNA species in nor-mal neurons that are contrasted by the pale backgroundof surrounding neuropil and white matter tracts thatlack abundant nucleic acids. In addition, RNA seques-tration has been found in senile plaques and neuronsthat bear neurofibrillary tangles (NFTs) in Alzheimer’sdisease (AD) using AO histofluorescence (11,17,18).

Accession of Single Cells from Tissue Sections

Microdissection of individual cells is performedto enable downstream gene expression profiling usingcDNA microarrays or by PCR-based technologies. Pro-vided that procedures are performed on well-preservedtissue sections and RNase-free conditions are em-ployed, both immunocytochemical and histochemicalprocedures can be utilized to identify specific cell(s)of interest. Several different methodologies have beenused to aspirate individual neurons or groups of cellsincluding single cell microaspiration and laser capture

microdissection (LCM) (1,4,6,19). Prior to discussingsingle cell microdissection, the merits of regional dis-sections are illustrated. Groups of related cells fromdiscrete regions of brain can be readily dissected fromfresh tissues, frozen tissue blocks, paraffin-embeddedfixed tissue sections (e.g., 5–6 mm thick), and/or cryo-protected, fixed frozen tissue sections (e.g., 20–40mmthick) by an experienced neuroanatomist or neuro-pathologist. In terms of tissue section preparation, un-stained sections can be utilized, but optimal cellularresolution occurs using sections prepared for immuno-cytochemistry or histochemistry. Regional dissectionscan be performed using a stereomicroscope and ascalpel blade or micropunch. RNA is then extractedfrom the desired tissue using Trizol reagent (Invitro-gen) or other suitable extraction solutions for down-stream applications such as cDNA array analysis,including studies of hippocampal cytoarchitecture (20),and disease pathogenesis in AD (21), amyotrophiclateral sclerosis (22–24), and schizophrenia (25–28),among others. Regional tissue dissections can also beutilized for additional downstream applications in-cluding library subtraction, real-time quantitative PCR(Q-PCR) and differential display PCR (29–33). The ad-vantage of regional analysis is that limited RNA am-plification is necessary to generate significant hybridi-zation signal intensity for cDNA arrays. The obviousdisadvantage of regional dissection procedures is thelack of single cell resolution, as neurons, noneuronalcells, vascular elements, and epithelial cells will be in-cluded in the dissection.

Advances in molecular techniques and functionalgenomics approaches allow scientists to extract nucleicacids from tissue sources for precise molecular analysis.The efficacy of sophisticated genetic testing methods isdependent upon the purity and precision of the analyzedcell population(s). Bulk homogenizing tissue samplesresults in the mixture of several different cell types.Cell-specific molecular analysis demands accurate,non-destructive isolation of cell populations from opti-mally prepared tissue sections. Two popular microdis-section methodologies are LCM and microaspiration.LCM is a widely used technique that was developed atthe NIH (34,35) and marketed through collaborationwith Arcturus (www.arctur.com). LCM employs a high-energy laser beam that enables thermal adhesion be-tween the desired cells and thermoplastic transfermaterial embedded on a microfuge tube cap. In thismanner, single cells as well as dozens to hundreds ofcells can be adhered onto the thermoplastic in a rela-tively short time. Moreover, RNA, DNA, and proteinextraction methods can be performed on microdissected

982 Ginsberg and Che

cells (36–39). However, LCM use is most noted forRNA extraction and subsequent application to cDNAmicroarrays. Microaspiration entails visualizing an in-dividual cell (or cells) using an inverted microscopeconnected to a micromanipulator, microcontrolled vac-uum source, and an imaging workstation on an air table.Electrophysiology rigs can also be modified to aspiratecells from fixed tissue sections with minor modifica-tions. Hand-held and syringe-pump driven vacuumsources can also be utilized, however, they are difficultto control and may cause inadvertent damage to the tis-sue section. Microaspiration results in accurate dissec-tion of the neurons of interest with minimal disruptionof the surrounding neuropil, including neurons fromnormal brains as well as from cells undergoing variousforms of neurodegeneration (7,10,12,40,41). Microaspi-ration can also be used to isolate individual dendritesand other perikaryal profiles for downstream analysis.Single cells can be used alone, or pooled with othercells for subsequent RNA amplification.

RNA Amplification of Single Cells andPopulations

cDNA array technology essentially allows theinvestigation of multiple (e.g., hundreds to thousands)genes simultaneously from one tissue sample. Compar-ing the qualitative and quantitative changes of multiplegenes assists in the process of elucidating molecularmechanisms of functional changes. However, fairlylarge quantities of tissues are needed for subsequentRNA extraction. Reverse transcription-polymerase chainreaction (RT-PCR) can amplify genetic signals fromsmall tissue samples, yet only one (or a few) genes canbe analyzed concurrently. PCR-based methods tend toamplify abundant genes over rare genes, and may dis-tort quantitative relationships among gene populationsdue to exponential, non-linear amplification (42,43).In contrast, in vitro RNA transcription using a doublestranded (ds) cDNA template can amplify genes in alinear manner (40,44,45). Therefore, this methodologypreserves the original quantitative relationship(s) in anamplified gene population, facilitating downstreamquantitative analysis.

Several RNA amplification procedures are cur-rently available. These methodologies effectively am-plify RNA from small samples, yet they are relativelydifficult protocols to perform for amplifying geneticsignals. The main obstacle for increasing the effi-ciency of the method is the problematic second strandcDNA synthesis. Two procedures currently in use for

second strand cDNA synthesis include self-primingand replacement synthesis. Self-priming utilizes thehairpin formed at the 39 end of first strand cDNA toself-prime the synthesis of second strand cDNA. How-ever, the loop formed at the end must be removedusing S1 nuclease digestion or related procedures, thatinevitably lead to the loss of the 59 signal (46). Inaddition, self-priming is performed with the Klenowfragment of E. coli DNA polymerase I, an enzymewith relatively low processivity, further decreasing theefficiency of the method. The second procedure cur-rently used for second strand cDNA synthesis, termedreplacement synthesis, avoids S1 nuclease digestionand has been used for RNA amplification (47). Thisreaction employs multiple enzymes to synthesize asecond strand cDNA including RNase H, DNA poly-merase I, and T4 DNA ligase. After the first strandcDNA is synthesized, it is annealed to the templateRNA strand. Replacement synthesis uses the activityof RNase H to produce multiple nicks in the RNAstrand. These nicks then serve as initiation sites forsecond strand cDNA synthesis by DNA polymerase I.Eventually, most of the RNA fragments are replacedby DNA. The ds cDNA molecules are finally treatedwith T4 DNA ligase to fill in any remaining nicks inthe second strand cDNA. However, replacement syn-thesis suffers from low efficiency, likely caused by themultiple enzymatic steps involved.

To enable a robust in vitro RNA transcription reac-tion for subsequent genetic analyses, a ds cDNA tem-plate requires a functional transcriptional promotersequence. Conventional RNA amplification proceduresutilize a bacteriophage transcriptional promoter se-quence (e.g., T7, T3, or SP6) to the 39 end of first strandcDNA, resulting in transcripts with antisense orienta-tion. Aspects of the current RNA amplification strategiesthat would benefit from innovative improvements in-clude increasing the efficiency of second strand cDNAsynthesis and allowing for flexibility in the placement ofbacteriophage transcriptional promoter sequences.

A new procedure has been developed that uses amethod of terminal continuation (TC) to attach anoligonucleotide primer of known sequence to the 59 re-gion of first strand cDNA (Figs. 1 and 2). Second strandcDNA synthesis is initiated by annealing a secondoligonucleotide primer complementary to the attachedoligonucleotide. By providing a sequence-specificprimer at the 39 region of first strand cDNA and a primer complementary to it, hairpin loops will not form, effectively obviating the need for S1 nuclease di-gestion. Second strand cDNA synthesis is performedusing Taq polymerase, and one round of amplification is

RNA Amplification in Brain Tissues 983

typically sufficient for downstream genetic analyses. Invitro transcription can be driven using a bacteriophagepromoter sequence attached to either the 39 primer or 59primer in the TC RNA amplification scheme (40).Therefore, orientation of amplified RNAs is either “anti-sense” or “sense,” depending upon the placement of thebacteriophage promoter sequence (Fig. 2).

The data presented herein illustrates the impor-tance of primer design and specificity for single cellRNA amplification. Data collected through the useof the TC RNA amplification are presented usinghuman hippocampal tissue sections from postmor-tem human brains and a mouse injury paradigm forsubsequent single cell analysis on custom-designedcDNA array platforms. Specifically, by varying thelength and nucleotide composition of the respectiveprimers, optimal RNA amplification is illustrated,effectively demonstrating the importance of consis-tent use of one primer set throughout an experimen-tal paradigm.

EXPERIMENTAL PROCEDURES

A schematic overview of the experimental paradigm is illus-trated in Figure 1. The diagram depicts the coordinated series of

steps ranging from accession of tissues and subsequent regional dis-section and/or single cell microaspiration through RNA amplifica-tion and downstream genetic analyses.

Postmortem Human Hippocampal Tissue Accession.Regionalhippocampal dissections and individual hippocampal neurons (CA1pyramidal neurons) are isolated from 6 mm-thick fixed (70% ethanolplus 150 mM sodium chloride or 4% paraformaldehyde) paraffin-embedded sections of hippocampus from neuropathologically con-firmed normal controls {Table 1; n 5 8; 4 male/4 female; mean age78.1 6 9.1 years; mean postmortem interval (PMI) 10.8 6 7.1 hours}as described previously (7,8,10). The brains used in this study areobtained from normal control subjects accessed through the ReligiousOrders Study, Rush-Presbyterian St. Luke’s Medical Center (see Ref.11) and the Center for Neurodegenerative Disease Research at theUniversity of Pennsylvania School of Medicine. All of the tissue sam-ples are harvested using the same methods and procedures. Each brainis confirmed to have abundant cytoplasmic RNAs by AO histofluo-rescence as described previously (17,18).

Mouse Dentate Gyrus Granule Cell Accession and Injury Par-adigm. Adult male C57BL/6 mice (15–30 grams) are used as naïvecontrols, or are subjected to sham surgeries, or occipital cortex (OC)lesions. Subjects reside in an animal room set on a 12-hour light/dark cycle with ad libitum access to food and water. The OC lesionparadigm serves as a surgical control for perforant path transectionstudies as described elsewhere for subsequent experiments (48). Allsurgical manipulations are performed as an aseptic procedure withminimal complications and morbidity. Mice are anesthetized with anintramuscular injection of ketamine (80 mg/kg of animal weight)followed by xylazine (13 mg/kg of animal weight). After any re-flexive movements have ceased, the animal is placed prone into astereotaxic apparatus using a mouse skull adapter (Stoelting) andtemperature maintained at 37°C with a heating pad. A 1.0 mm cran-iotomy centered approximately 6.5 mm caudal to bregma and 2.0 mm


Fig. 1. Flow diagram illustrating the experimental design for re-gional and single cell analysis using TC RNA amplification coupledwith custom-designed cDNA array technology.

Fig. 2. Schematic of the TC RNA amplification method. A. TC andpoly d(T) primers are combined with the mRNAs to be amplified(rippled line). Antisense orientation is obtained using a poly d(T)primer with an attached bacteriophage transcription promoter. Firststrand synthesis commences with the formation of an mRNA-cDNAhybrid following reverse transcription and terminal continuation ofthe oligonucleotide primers. RNase H digestion proceeds to removethe original mRNA template strand and second strand synthesis isperformed using Taq polymerase. The resultant double strandedcDNA is utilized as template for in vitro transcription, yielding highfidelity, linear RNA amplification of antisense orientation (rippledlines). B. Schematic similar to A, illustrating the TC RNAamplification procedure amplifying RNA in the sense orientation byattaching the bacteriophage transcription promoter to the TC primer.

lateral to the midline is made with a high speed dental drill andtrephine bit. The dura is incised with a sharp 25 gauge needle, ex-posing the cortical mantle. The OC lesion is performed by aspirationof the overlying occipital cortex and white matter with a blunt-tipped 22-gauge needle connected to a vacuum line, without dam-aging the angular bundle or subcortical structures (49–51). A shamlesion consists of a reflection of the dura following a craniotomy atthe same stereotaxic coordinates as the OC lesion. Once a lesion iscomplete, the skull is sealed with bone wax, and the fascia and scalpare closed in a single layer using an autoclip. Animals are placingtheir home cage for postoperative recovery (two or three animals percage) immediately following the surgical procedures. Mice aregiven an overdose of ketamine and xylazine and perfused transcar-dially with ice-cold 4% paraformaldehyde in phosphate buffer at theappropriate postlesion time point. Tissue blocks containing the dor-sal hippocampus are paraffin embedded. Six mm-thick tissue sec-tions are cut in the coronal or horizontal plane on a rotary microtomefor immunocytochemistry and histochemical procedures. Tissue sec-tions throughout the rostrocaudal extent of the dentate gyrus areprocessed for immunocytochemistry using several antibodies in-cluding nonphosphorylated neurofilament proteins (RMdO20; 52) todelineate granule cells and hippocampal lamination patterns, andMAP2 (Sigma) to identify granule cell dendrites in the inner molec-ular layer and outer molecular layer of the dentate gyrus. Deparaf-finized tissue sections are blocked in a 0.1 M Tris (pH 7.6) solutioncontaining 2% donor horse serum (DHS) and 0.01% Triton X-100for 1 hour and then incubated with primary antibodies in a 0.1 MTris/2% DHS solution overnight at 4°C in a humidified chamber.Sections are processed with the ABC kit (Vector Labs, Burlingame,CA) and developed with 0.05% diaminobenzidine, 0.03% hydrogenperoxide, and 0.01 M imidazole in Tris buffer for 10 minutes as de-scribed previously (7,8,51). Tissue sections are not coverslipped orcounterstained and are immersed in RNase-free 0.1 M Tris for micro-aspiration and subsequent TC RNA amplification.

TC RNA Amplification.Dissected hippocampal regions andindividual hippocampal neurons are incubated in 250 ml of pro-teinase K solution (Ambion, 50 mg/ml) for 12 hours at 37°C prior toextraction. RNA is either extracted using a conventional organicmethodology including Trizol reagent (Invitrogen) followed byphenol:chloroform extraction, or by a semi-automated magnetic ex-traction method using the KingFisher instrumentation (ThermoLabSystems) (53). TC RNA amplification entails synthesizing firststrand cDNA complementary to the RNA template, generating sec-ond strand cDNA complementary to the first strand cDNA, and

finally in vitro transcription using the ds cDNA as template. Twooligonucleotide primers, a poly d(T) primer and a TC primer, areemployed. The TC primer consists of two domains, a deoxyoligonu-cleotide sequence at the 59 terminus and a short span of three dCTPsor dGTPs at the 39 terminus. For antisense RNA amplification, thebacteriophage promoter sequence is placed on the poly d(T) primer(Fig. 2A). For the novel sense orientation, the bacteriophage se-quence is attached to the TC primer (Fig. 2B). TC and poly d(T)primer sequences are depicted in Table 2.

RNAs are reverse transcribed in the presence of the poly d(T)primer (50 ng/ml) and TC primer (50 ng/ml) in 1X first strand buffer(Invitrogen), 1 mM dNTPs, 5 mM DTT, 20 U of RNase inhibitor(Ambion), and 5 U reverse transcriptase (Superscript II; Invitrogen)in a final volume of 20 ml. The synthesized single stranded cDNAsare converted into ds cDNAs by adding into the reverse transcriptionreaction the following: 10 mM Tris (pH 8.3), 50 mM KCl, 1.5 mMMgCl2, 0.5 U RNase H (Invitrogen), and 5 U Taq polymerase (PEBiosystems) in a total volume of 100 ml. The samples are placed in athermal cycler and second strand synthesis proceeds as follows:RNase H digestion at 37°C, 10 min; denaturation at 95°C, 3 min, an-nealing at 50°C, 3 min; elongation at 75°C, 30 min. The reaction isterminated with 5 M ammonium acetate. The samples are then ex-tracted in phenol:chloroform:isoamyl alcohol (25:24:1) and ethanolprecipitated with 20 mg of linear acrylamide (Ambion) as a carrier.The solution is centrifuged at 14,000 rpm for 15 min and the pellet iswashed once with 95% ethanol and air-dried. The cDNAs are resus-pended in 20 ml of RNase free H2O and drop dialyzed on 0.025 mmfilter membranes (Millipore) against 50 ml of RNase-free H2O for2 hours. The sample is collected off the dialysis membrane and hy-bridization probes are synthesized by in vitro transcription using33P incorporation in 40 mM Tris (pH 7.5), 7 mM MgCl2, 10 mM NaCl,2 mM spermidine, 5 mM of DTT, 0.5 mM of ATP, GTP, and CTP,10 mM of cold UTP, 20 U of RNase inhibitor, and 40 mCi of 33P-UTP(Amersham Biosciences). The reaction is performed at 37°C for4 hours. Radiolabeled TC RNA probes are hybridized to custom-designed cDNA arrays without further purification.


Table 1. Case Demographics

Case # Gender Age PMI Fixation

1 F 92 9 ETOH2 F 83 22 ETOH3 M 65 26 ETOH4 M 75 7 ETOH5 F 74 3.5 ETOH6 M 69 3 PARA7 F 80 12 PARA8 M 87 4 PARA

Abbreviations: ETOH, 70% ethanol buffered with 150 mM sodiumchloride; PARA, 4% paraformaldehyde; PMI, postmortem interval.Age is reported in years and PMI is reported in hours.

Table 2. Primer Composition

Primer Basepair Compositions Basepairs

poly d(T)-T7 3´-AAA CGA CGG CCA GTG 66AAT TGT AAT ACG ACT CACTAT AGG CGC TTT TTT TTT

TTT TTT TTT TTT TTT-5´TC primer (C) 5´-TAT CAA CGC AGA 17

GTC CC-3´TC primer (G) 5´-TAT CAA CGC AGA 17

GTG GG-3´TC primer (A) 5´-TAT CAA CGC AGA 17

GTA AA-3´TC primer (T) 5´-TAT CAA CGC AGA 17

GTT TT-3´TC primer 5´-TAT CAA CGC AGA GTN 23

N(6)C NNN NNC CC-3´TC primer 5´-TAT CAA CGC AGA GTN 23

N(6)G NNN NNG GG-3´poly d(T) 3´-TTT TTT TTT TTT TTT TTT-5´ 18TC-T7 primer 5´-AAA CGA CGG CCA GTG 51

AAT TGT AAT ACG ACT CACTAT AGG CGC GAG AGC CCC-3´

Custom-Designed cDNA Arrays.Array platforms consist of 1 mgof linearized cDNA purified from plasmid preparations adhered tohigh-density nitrocellulose (Amersham Biosciences). Each cDNAand/or expressed sequence-tagged cDNA (EST) is verified by se-quence analysis and restriction digestion. cDNA clones and ESTsfrom mouse, rat, and human are employed. Arrays are prehybridized(12 hours) and hybridized (48 hours) in a solution consisting of 63

SSPE, 53 Denhardt’s solution, 50% formamide, 0.1% sodium do-decyl sulfate (SDS), and denatured salmon sperm DNA (200 mg/ml)at 42°C in a rotisserie oven (7,8). Following hybridization, arraysare washed sequentially in 23 SSC/0.1% SDS, 0.53 SSC/0.1%SDS, and 0.13 SSC/0.1% SDS solutions for 20 minutes each at42°C. Arrays are placed in a phosphor screen for 48 hours and de-veloped on a PhosphorImager (Amersham Biosciences).

Data Analysis.Hybridization signal intensity is quantified bysubtracting background using empty vector (pBs). Expression ofTC amplified RNA bound to each linearized cDNA (approximately250 cDNAs/ESTs) is expressed as a ratio of the total hybridizationsignal intensity of the array, thereby minimizing variations due todifferences in the specific activity of the probe and the absolutequantity of probe present (4). Data analyzed in this manner does notallow the absolute quantitation of mRNA levels. However, an ex-pression profile of relative changes in mRNA levels is generated.Relative changes in individual mRNAs are analyzed by one-wayanalysis of variance (ANOVA) with post-hoc analysis (Newman-Keuls test) for individual comparisons. Differentially expressedgenes are also clustered into functional protein categories for multi-variate coordinate gene expression analysis.

RESULTS

TC Primer Composition Is Critical for RNAAmplification

TC RNA amplified transcript yield and size distri-bution profiles are estimated by capillary gel electro-phoresis (Bionanalyzer 2100, Agilent Technologies) inconjunction with a RNA6000 LabChip (Ambion). Theassay combines the use of a capillary device with fluo-


Fig. 3. Electropherogram of TC RNA amplification products usingdifferent TC primers. Note the long-range representation of transcriptsfrom high (greater than 7.5 Kb) to low (hundred bp) on the gel. TCRNA amplification is performed on adjacent regional hippocampaldissections using the poly d(T)-T7 primer, and varying the TC primer.Lane 1 depicts the molecular weight standards. Lane 2 is a control laneof sample without TC and poly d(T) primers. Lanes 3 and 4 illustraterobust RNA amplification with TC primers containing C or G at the 3´terminus. Lanes 5 and 6 illustrate a lack of RNA amplification with TCprimers containing A or T at the 3´ terminus. Lanes 7 and 8demonstrate that random addition of basepairs (N) leads to RNAamplification with an abundance of small molecular weight fragmentsthat are not found in lanes 3 and 4.

Fig. 4. cDNA array analysis of postmortem hippocampus followingTC RNA amplification with TC primers of differing nucleotidecomposition. Representative custom-designed cDNA arrays illustrat-ing relative expression levels of eight transcripts {top array labeled 1-8 is the key; cyclic AMP response element binding protein (CREB),cyclin D1 (cycD1), immediate early gene zif268, nuclear factor-kappaB (NFKb), neural cell adhesion molecule (NCAM), cyclooxygenase-2(COX-2), and galanin receptor 1 & 2 (GalR1, GalR2). The secondarray is a control of sample without the TC and poly d(T) primers. Thethird and fourth arrays illustrate varying levels of hybridization signalintensity for the eight transcripts using TC primers containing C or Gat the 3´ terminus, respectively. In contrast, the fifth and sixth arraysillustrate a paucity of hybridization signal intensity using TC primerscontaining A or T at the 3´ terminus, respectively.

Fig. 5. Single cell cDNA analysis of CA1 pyramidal neurons usingcustom-designed cDNA arrays and TC RNA amplification.Representative arrays illustrate a wide dynamic range ofhybridization signal intensities for the eight cases utilized in thestudy (see Table 1). The negative control (neg) is a single CA1pyramidal neuron from case #1 that does not have the primersnecessary for TC RNA amplification. In addition, a moderatevariation of gene level expression across the eight human cases isalso observed, indicating the utility of using postmortem humansamples for normative and neuropathological investigations. Key:notch, amyloid-ß precursor-like proteins 1&2 (APLP1, APLP2),amyloid-ß precursor protein (APP), cathepsin D (catD), heat shockprotein 60&70 (HSP60, HSP70), heparan sulfate proteoglycan(HSPG), arc, ubiquitin (ubiq), glutaredoxin (GRX), and superoxidedismutase1 (SOD1).

rescent RNA dye chemistry for the electrophoretic sep-aration and detection of RNA transcripts. For example,previous bioanalysis assessment of TC RNA amplifi-cation efficiency (40) has demonstrated an approximate1000–1500 fold amplification using post mortem hip-pocampus (from the same cases as the present study) asstarting material. Results of the present study indicatethat TC RNA primer composition is a critical compo-nent of the efficiency of TC RNA amplification (Figs.3 and 4). Specifically, antisense (e.g., the bacterio-phage promoter sequence is attached to the 39 poly d(T)primer, Table 2) TC RNA amplification is performedon adjacent regional hippocampal dissections using thepoly d(T)-T7 primer, and varying the TC primer. TCprimers containing C or G at the 39 terminus exhibit ro-bust activity, whereas TC primers containing A or T atthe 39 terminus do not amplify RNA above control lev-els (sample amplified without primers) (Figs. 3 and 4).Random addition of basepairs (N) leads to RNA am-plification for TC primers using C or G. However, aprofuse increase in small molecular weight “oligo-like”fragments is observed on the electropherogram (Fig. 3)and an increased level of background hybridizationusing custom-designed cDNA arrays (data not shown).Based upon these results, optimal TC RNA amplifica-tion occurs using TC primers with C or G content, andhas strong implications towards the mechanism of ac-tion of the TC primer system.

Expression Profiles of Single CA1 Neurons UsingTC RNA Amplification

TC RNA amplification is coupled with single cellmicroaspiration to develop a series of molecular finger-prints for human hippocampal neuronal subtypes. Anti-sense TC RNA amplification {poly d(T)-T7 primer andTC (C) primer} using individual neurofilament-immunoreactive CA1 pyramidal neurons (4,7) is per-formed in conjunction with custom-designed cDNAarrays. Representative gene expression within individ-ual CA1 pyramidal neurons is illustrated in Figure 5.A wide dynamic range of varying levels of hybridiza-tion signal intensity is observed for individual cDNAs,including high [e.g., cathepsin D (catD), heparan sulfateproteoglycan (HSPG), arc, and glutaredoxin (GRX)},moderate {notch, amyloid-b precursor protein (APP),ubiquitin (ubiq), and superoxide dismutase1 (SOD1)],and low {amyloid-b precursor-like proteins 1&2 (APLP1, APLP2), and heat shock protein 60&70 (HSP60,HSP70)}. Variation of gene level expression acrossthe eight human cases is also represented by custom-designed cDNA arrays (Fig. 5) to illustrate the moder-

ate differences in hybridization signal intensity levelsfor the twelve depicted transcripts. These data providea normative database in nondemented control brainsthat will continue to be used to probe for neuropatho-logical changes in expression levels of single neuronalpopulations in the brains of subjects with neurologicalor neuropsychiatric disorders.

Molecular Fingerprints of Dentate Gyrus GranuleCells

TC RNA amplification methodology is combinedwith custom-designed (.250 cDNAs/ESTs) cDNAarrays to generate an expression profile of dentate gyrusgranule cells undergoing injury and reactive synaptoge-nesis following axotomy. The expression profile of sev-eral classes of relevant transcripts is being evaluated,including glutamate receptors (GluRs), glutamate re-ceptor interacting proteins, synaptic-related markers,and AD-related genes. Preliminary results indicatea significant down regulation of AMPA (GluR1 andGluR2) and kainate [KA; (GluR6 and GluR7)] receptorsubunits following both perforant path transections and


KA injections (4,54). These data are consistent with aninitial excitotoxic mechanism underlying part of the cel-lular alterations following perforant path transections(48). OC lesions are employed in the current study as alesion control paradigm, as the overlying OC must beaspirated when performing perforant path transectionsin order to visualize the angular bundle where the per-forant path resides at this rostrocaudal level. We hy-pothesize that OC lesions will not alter the regulation ofGluRs and other transcripts in dentate gyrus granulecells since the principal glutamatergic input (i.e., theperforant path) is not damaged by the lesion.

Pooling 10 and 25 individually aspirated cells persection for RNA amplification and subsequent cDNAarray is evaluated along with single granule cells (Fig. 6).The pooling procedure is initiated because of greatervariability and lower signal detection from one gran-ule cell, likely due to their small size (approximately5–8 mm in diameter) and high packing density withinthe granule cell layer. Both 10 and 25 cell pools gen-erate robust hybridization signal intensity compared tosingle granule cells. The 10 cell pool is utilized forsubsequent cDNA array analysis of gene expression ingranule cells following OC lesions and sham surgeries.Results indicate that AMPA and KA receptor expres-sion levels are not altered on either the side ipsilateralor side contralateral to the lesion as compared to naïvecontrols at six postlesion time points (1, 5, 10, 14, 30,and 60 days postlesion) as illustrated by representativecustom-designed cDNA arrays (Fig. 7; 14 days postle-

sion is depicted). Both GluR1 and GluR2 are ex-pressed at high levels within granule cells, whereasmoderate to low levels of GluR4, and low levels of


Fig. 6. Arrays illustrating the relative hybridization signal intensityof several transcripts from individual and pooled mouse dentategyrus granule cells. Moderate and high levels of ß-tubulin (b-tub) andlow molecular weight neurofilament protein (NF-L) are observed,respectively, in single cells along with very low to undetectablelevels of three repeat tau (3tau) and low to moderate levels of fourrepeat tau (4tau). Both 10 and 25 cell pools generate robusthybridization signal intensity compared to single granule cells.

Fig. 7. Lack of regulation of AMPA receptors GluR1-GluR4 inmouse dentate gyrus granule cells following unilateral OC lesionsand sham surgeries using TC RNA amplification coupled withcustom-designed cDNA arrays. A. Representative arrays illustratingno significant changes in GluR1, GluR2, GluR3, and GluR4expression on sides ipsilateral (ipsi) and contralateral (contra) to OClesions and sham surgeries as compared to naïve controls. B.Histogram demonstrating the relative hybridization signal intensitylevels for the AMPA receptors. C. Histogram demonstrating noquantitative differences between the sides ipsilateral andcontralateral to the lesions as compared to control (demarcated as100%) for GluR1-GluR4.

GluR3 are observed (Fig. 7B). Further, KA receptorsGluR6 and GluR7 are expressed at high levels withinindividual pools of granule cells. Hybridization signalintensity levels for GluR6 and GluR7 do not vary fol-lowing OC lesions or sham surgeries (Fig. 8).

DISCUSSION

cDNA Microarray Technology

Recent technical advances have fostered thedevelopment of high-density cDNA microarrays thatenable high throughput analysis of hundreds to thou-sands of genes simultaneously. Synthesis of cDNAmicroarrays entails adhering cDNAs or ESTs to solidsupports such as glass slides or nylon membranes(55,56). A parallel technology uses photolithographyto adhere oligonucleotides to array media (57). Geneexpression is assayed by harvesting total RNA ormRNA from sample tissues, labeling either by ra-dioactive, biotinylated, or fluorescent methods, andthen hybridizing the probes to cDNA arrays. Arraysare washed to remove nonspecific background hy-bridization, and imaged using a phosphor imager forradioactively labeled probes and by a laser scanner forfluorescently labeled probes. Gene expression is thenquantitated using informatics software that enableslarge volumes of coordinate analyses. Computationalanalysis is critical for optimal use of cDNA microar-rays due to the enormous volume of data that is gener-ated from a single probe. Additionally, access torelational databases is desirable, especially when eval-uating hundreds of ESTs that may/may not be linked togenes of known function. Although cDNA microarraytechnology has great appeal for many biological sys-tems, relatively little has been published so far in thebrain (6,58), in part due to the heterogeneity of brainregions and cell types. Therefore, the combination of

cDNA microarray technology with single cell analysisis a highly desirable paradigm whereby expressionprofiles of single populations of neuronal and noneu-ronal subtypes can be analyzed and compared undernormal and pathological conditions.

TC RNA Amplification Methodology

Gene profiling is a powerful tool to examine the ex-pression of multiple genes simultaneously. This para-digm can provide valuable insight into thepathophysiology of disease, tools for diagnosis, andguidance for the development of new pharmacothera-peutic interventions. However, one significant obstaclefor the most effective application of gene profiling tech-nology is the relative difficulty in utilizing small samplesfor subsequent downstream genetic analysis. The devel-opment of techniques such as LCM (34,35) and singlecell microaspiration (4,5) has allowed for the accessionof minute amounts of starting materials including singlecells as well as clusters of homogeneous cells in vitroand in vivo. However, RNA amplification is necessaryto generate sufficient hybridization signal intensity fordetection on cDNA microarrays. PCR-based strategiesare not ideal for this application because exponentialamplification does not preserve quantitative relation-ships between variably expressed genes at the same levelas linear RNA amplification. Hybrid protocols combin-ing PCR with RNA amplification have been employedsuccessfully for cDNA microarray analysis (59).

The success of the TC RNA amplification method(or virtually any molecular-based amplification proce-dure) is predicated on optimal preservation of RNAspecies in the tissues to be studied. A virtual revolu-tion has occurred in the ability to mine RNA data fromfixed tissues, provided proper RNase-free conditionsare employed (1,4). A critical component of the TCRNA amplification method is the highly efficient sec-ond strand cDNA synthesis. Traditionally, this step isinefficient when the 59 sequence of the first strandcDNA is not known, as sequence-specific primers can-not be generated to prime the second strand synthesis.In contrast, the TC method attaches an oligonucleotideprimer of known sequence (e.g., Table 2) to 39 termi-nus of the synthesized first strand cDNA, thus provid-ing a molecular scaffolding for the priming of thesecond strand synthesis. Essential structural require-ments include a short stretch of C or G at the 39 ter-minus of the TC primer. Replacement of C/G with Aor T vastly diminishes the efficiency of TC RNA am-plification (Figs. 3 and 4).

The mechanism underlying the activity of the TC


Fig. 8. Expression of KA receptors GluR6 and GluR7 following OClesions and sham surgeries. No significant changes are observed onthe sides ipsilateral or contralateral to OC lesions or sham surgeriesas compared to controls (left panel). A moderate expression level ofGluR6 and GluR7 is illustrated in a histogram (right panel).

primer system is being evaluated experimentally. TheTC primer must base pair with complementary C’s orG’s at the termination site of the reverse transcriptionreaction in order to prime cDNA synthesis. Severalpotential locations have been implicated for this com-plementary interaction to occur. For example, the re-verse transcriptase reaction will add a few C’snonspecifically at the end of mRNA template (60), andwe have demonstrated that both C’s and G’s are addedby reverse transcriptase activity that may base pair withthe TC primer. A proposed mechanism of TC activitymay involve interactions with CG islands (CpG islands).CpG islands are short, variable nonmethylated regionsof CG-rich sequences prevalent at the 59 region of ap-proximately 60% of all human genes, and are found at asignificantly less frequency (CpGs are 25% less fre-quent than predicted) throughout the rest of the genome(61–63). CpG islands may represent a site whereby TCprimers preferentially anneal, and can account for thelong transcripts that are synthesized during the TC RNAamplification procedure (see Fig. 3). A caveat of the TCRNA amplification method is that the composition ofthe TC primer determines the efficacy of amplification.Therefore, it is important to use same primer (e.g., C orG) consistently (40) in any given study.

Strategies for cDNA Microarray Verification

Independent verification of individual gene levelchanges discovered by cDNA array analysis by alter-nate techniques is of critical importance. However,many current techniques are fraught with methodolog-ical considerations that have to be appreciated whenapplying the respective assay(s). For example, PCR-based analyses are exponential, rather than linear analy-ses. Therefore, following the initial cDNA synthesisstep by reverse transcriptase, subsequent amplificationby PCR is exponential. Unless careful quantitativeRT-PCR is performed, the PCR amplification step caninduce significant cell to cell variability (1,42). In ad-dition, to attain the necessary level of sensitivity formessages of low abundance, a second round of PCRwith internal nested primers is performed. The dis-advantage of this approach is the high risk of contam-ination and the small number of genes that can beanalyzed simultaneously. In contrast, real-time Q-PCRlessens concerns regarding linear amplification, as di-rect detection of PCR products occurs during the ex-ponential phase of the reaction, combining amplificationand detection in a single step. Other advantages ofreal-time Q-PCR include high throughput capabilities,the ability to simultaneously multiplex reactions, en-

hanced sensitivity, reduced inter assay variation, andlack of post-PCR manipulations. A relative disadvan-tage of Q-PCR is that the apparatus and the fluorescentprobe sets necessary for the assay are expensive.

In situ hybridization histochemistry is an extremelyuseful technique, especially when applied to well-processed brain sections, allowing for the localizationand estimation of abundance of specific mRNA tran-scripts within single cells (64). Quantitation of reac-tion product density in the case of non-radioactiveprobes such as digoxigenin, or densitometry of silvergrains from radiolabeled probes, can be applied. Dueto several technical considerations including limitedsensitivity and relative inability to assess multiplemRNAs simultaneously, in situ hybridization is bestsuited for regional assessments of gene expression pat-terns, or as a companion technique to PCR- or linear-based single cell analyses.

Further, it is highly desirable to supplement arraydata with measurements of protein expression by im-munoblotting, immunocytochemical, ELISA, or pro-teomic techniques. In terms of protein assessments,single cell resolution can be attained in tissue sectionsusing immunocytochemical methods. However, im-munoblotting and proteomic-based procedures at pres-ent cannot (at least in vivo) be performed on single cellsand must rely on tissue homogenate preparations. Thus,a combination of multidisciplinary approaches is idealfor verification of gene expression level alterations,with the explicit knowledge that the sum of the evalua-tions may be more informative and reflect the actual bi-ology of the system than an individual method.

CONCLUSIONS

The combination of single cell microdissection,TC RNA amplification, and cDNA microarrays enableshigh resolution, high throughput expression profilingof hundreds to thousands of genes simultaneously froma single neuron or a neuronal population. The nextlevel of understanding of cellular and molecular mech-anisms underlying normative function and the patho-physiology of disease lies in the ability to combinethese aforementioned technologies with appropriatemodels to recapitulate the structure and connectivity ofthese complex systems in vivo and in vitro. For exam-ple, lesion-induced injury paradigms, transgenic animaldevelopment, and cultures of human-derived neuronsand stem cells are likely to be the substrates for re-gional and single cell investigations using cDNA arraytechnology. Ultimately, however, these analyses mustbe brought into the context of human neurobiology and


neuropathology for downstream drug discovery andpharmacotherapeutic interventions.

ACKNOWLEDGMENTS

We thank Mr. John T. Le and Ms. Irina Elarova for expert tech-nical assistance. Support for this project (SDG) comes from theNINDS (NS43939), NIA (AG10668, AG17617, and AG14449) andAlzheimer’s Association. We also express our appreciation to thefamilies of the patients studied here who made this research possible.

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RNA Amplification in Brain Tissues