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798 NATURE BIOTECHNOLOGY VOL 17 AUGUST 1999 http://biotech.nature.com

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Methods for gene expression analysis include transcript sampling bysequencing1–3 or hybridization signature14–17, transcript amplifica-tion and imaging4–8, and hybridization to gene arrays9–13. Serialanalysis of gene expression (SAGE)2, the most cost-effective method,is limited by the small amount of sequence information obtained foreach gene. Transcript sequencing following subtractive hybridiza-tion is limited to binary comparisons3. Transcript imagingapproaches such as differential display4, partitioning by type IISrestriction enzymes6, representational difference analysis (RDA)7,and amplified fragment length polymorphism (AFLP)8 are rapid andcomprehensive (in theory) since they use fragment patterns on gelsto infer gene expression. However, they require a time-consumingcloning and confirmation process to identify differentially expressedgene fragments. The development of microarrays has significantlyenhanced the capacity of hybridization techniques to identify differ-ences in gene expression9–13. In practice, however, hybridizationmethods are limited by an inability to detect genes with no expressedsequence tag (EST) representation.

We describe here a novel approach to expression analysis thatprovides rapid, comprehensive sampling of cDNA populationstogether with sensitive detection of differences in mRNA abundancefor both known and novel genes. Using this method, we have ana-lyzed gene expression in a rat model of pressure overload-inducedcardiac hypertrophy.

Results and discussionThe method comprises three steps: restriction endonuclease diges-tion, adaptor ligation, and PCR amplification. Following double-stranded cDNA synthesis of poly-A+ RNA, cDNA pools are digestedwith different pairs of restriction enzymes with 6-bp recognitionsites. Complementary adapters are ligated to the digested cDNA, andadapter-specific primers are used to direct 20 cycles of PCR. Oneadapter-specific primer is biotin-labeled, while the other is labeledwith the fluorescent dye fluorophore fluorescamine (FAM).Following PCR amplification, the biotin-labeled DNA is purified on

immobilized streptavidin. Denatured single-stranded DNA frag-ments are electrophoresed on ultrathin polyacrylamide gels, andFAM-labeled fragments are detected by laser excitation. Since thebiotin label is necessary for purification and the FAM label is neces-sary for detection, all detected fragments result from restrictiondigestion with both enzymes. Typically 48–96 reactions are per-formed, each with a separate pair of endonucleases.

Identification of differential gene expression. Three indepen-dent reactions from the same cDNA sample are compared for quali-ty of electrophoretic peak resolution and reproducibility of peak pat-terns (Fig. 1A). Composite traces from each sample are generatedand compared (Fig. 1B). A composite trace is calculated for eachsample group based on average peak height and variance, and com-pared among sample groups using software designed to identifypeaks representing differential expression. cDNA fragments repre-senting differentially expressed genes are identified by comparingthe length of each fragment against a database search. Fragments ofspecific predicted lengths allow immediate identification of geneswhose sequences are in the database. Fragments derived from novelgenes are flagged by their absence from the database. Given a three-nucleotide size window, database look-up can provide a uniqueassignment of gene identity. The detection of multiple fragmentsderived from a single gene increases the likelihood that the predic-tion of the gene identity is correct (Fig. 1C).

Independent confirmation of gene identity. A method for rapidconfirmation of the identity of DNA fragments determined by data-base searching was developed. The reaction containing the fragmentof interest is performed a second time using the same primers but inthe absence and presence of an excess of an unlabeled oligonucleotidewhose sequence is derived from the predicted gene fragment. If thefragment identity was predicted correctly, the unlabeled oligonu-cleotide will compete for the universal oligonucleotide and reduce thecorresponding peak without affecting the amplification of the otherfragments (Fig. 1D). In addition to confirming the identity of a gene,this method allows more precise estimation of the magnitude of

Gene expression analysis by transcriptprofiling coupled to a gene database query

Richard A. Shimkets1*† , David G. Lowe2† , Julie Tsu-Ning Tai2,3, Patricia Sehl3, Hongkui Jin2, Renhui Yang2,Paul F. Predki1, Bonnie E. G. Rothberg1, Michael T. Murtha1, Matthew E. Roth1, Suresh G. Shenoy1, AndreasWindemuth1, John W. Simpson1, Jan F. Simons1, Michael P. Daley1, Steven A. Gold1, Michael P. McKenna1,

Kenneth Hillan2, Gregory T. Went1, and Jonathan M. Rothberg1

CuraGen Corporation, 555 Long Wharf Drive, New Haven, CT 06511. 2Departments of Cardiovascular Research and Pathology, Genentech, Inc., 1 DNA Way, SouthSan Francisco, CA 94080. 3Current address: CVTherapeutics, 3172 Porter Dr., Palo Alto, CA 94304. †Both authors contributed equally to this work.

*Corresponding author (e-mail: [email protected])

Received 1 July 1998; accepted 24 May 1999

We describe an mRNA profiling technique for determining differential gene expression that utilizes, butdoes not require, prior knowledge of gene sequences. This method permits high-throughput reproducibledetection of most expressed sequences with a sensitivity of greater than 1 part in 100,000. Gene identifi-cation by database query of a restriction endonuclease fingerprint, confirmed by competitive PCR usinggene-specific oligonucleotides, facilitates gene discovery by minimizing isolation procedures. Thisprocess, called GeneCalling, was validated by analysis of the gene expression profiles of normal andhypertrophic rat hearts following in vivo pressure overload.

Keywords: cardiac hypertrophy, differential gene expression, transcript profiling, oligonucleotide poisoning1

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NATURE BIOTECHNOLOGY VOL 17 AUGUST 1999 http://biotech.nature.com 799

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expression differences. Oligonucleotide competition is also useful forthe isolation of novel genes, as custom oligonucleotides can bedesigned against each distinct gene sequence recovered.

Coverage of cDNA sequences. The extent to which expressedsequences are sampled was tested in computer simulations usingnonredundant cDNA sequence databases containing a minimum of5,000 distinct gene sequences, including the TIGR human database,Genbank yeast, and Genbank corn databases. The coverage was cal-culated based on the generation of fragments <1,000 bp from a sim-ulated database restriction digest. Analysis of the yeast, human,mouse, and corn databases revealed that cleavage with 72 enzymepairs will yield at least one detectable cDNA fragment from morethan 90% of expressed genes and an average of 2.3 fragments pergene (Fig. 2A).

To assess the capacity to accurately digest fragments of DNA,amplify those fragments, correctly purify fragments with differentend sequences, and resolve them to their correct lengths, the methodwas performed using 10 enzyme pairs on Haemophilus influenzaegenomic DNA, for which the entire genomic sequence (1.4 Mb) isknown (Fig. 2B). Of a total of 330 DNA fragments predicted, 325fragments were detected within 1.5 nucleotides of the predictedmobility (Fig. 2C). One fragment was detected that had not been

predicted. Upon sequencing, this fragment was found to represent asequence polymorphism in the H. influenzae genome.

Reproducibility, sensitivity, and false-positive rate. Expressionprofiles were compared from a single cDNA synthesis, from differ-ent cDNA syntheses and between treatment groups. Repeated sin-gle reactions from the same cDNA had a variance in peak height ofabout 20%, with the variance dependent on fragment intensity(Fig. 3A). Comparing three replicate reactions from the samecDNA pool to three replicates from another cDNA pool of liketreatment improved the correlation to a variance of less than 5%(Fig. 3B), which enabled clear identification of differences whenthree composites of one treatment type were compared to threecomposites of another treatment type (Fig. 3C). In practice, thelatter experimental design is always used. To determine the rangeof mRNA abundance detected, plasmid DNA (hereafter referred toas “dope”) was introduced into rat liver cDNA at concentrations of1:5,000, 1:25,000, and 1:125,000 copies (Fig. 4A,B). Defined peaksfor each doped fragment were present against a constant back-ground, demonstrating that this process could resolve fragmentsspanning the range of expression levels seen in most cell types.

To test whether biases in the production of double-strandedcDNA from total RNA might be introduced that affect the sensitivityor reproducibility of the technique, a set of RNAs from human geneswas doped into RNA from HeLa cells at four different concentra-tions ranging from 1:1,300 to 1:130,000 (Fig. 4C). Of the 14 frag-ments generated from the chemistry protocol, 12 (86%) were abovebackground at 1:13,000, eight (57%) were detected at 1:65,000, and

Figure 1. Generation of GeneCalling profiles. (A) Trace replication ofseparate reactions run from the same rat heart sample. A portion of theelectrophoresis output for a set of triplicate amplification reactionsfrom a single sample is shown. The expression profile of each tissuesample is then generated as a composite of at least three reactions. (B)Sample replication of composite traces from different tissue samples.Reproducibility in the expression profile between different animals isillustrated for three different rat heart samples. (C) Examples ofmultiple band differences corresponding to the same gene. Three ofthe differences due to induction of biglycan in POL compared to sham-treated hearts are depicted. The labels m0, i0, n0, p0, and e1 arearbitrary symbols for the restriction enzymes used. (D) Oligonucleotidepoisoning reactions. The green and red traces were generated byreamplification without the addition of the gene-specificoligonucleotide, while the black and blue traces were generated byreamplification in the presence of the gene-specific primer.

Figure 2. GeneCalling coverage. (A) Examination of nonredundantcDNA sequence databases for yeast, human, mouse, and cornrevealed that cleavage with 72 enzyme pairs will yield at least onedetectable fragment <1 kb from >90% of expressed genes,independent of mRNA complexity. (B) Comparison of predicted(vertical black lines) to detected fragments resulting from arestriction digest performed on Haemophilus influenzae genomicDNA. Points on the axes indicate a predicted band that was notdetected or a detected band that was not predicted. (C) Correlationplot of detected versus predicted lengths of the fragments generatedby digestion of the H. influenzae genome with 10 restriction enzymepairs. Points on the axes indicate either a predicted band that wascompletely absent or a detected band that was not predicted.

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seven (50%) were detected at 1:130,000. Different fragments presentat the same concentration demonstrated different peak intensity, butamplification of the same fragment at different concentrationsdemonstrated roughly linear intensity when plotted against concen-tration (Fig. 4C). Liver cDNA prepared from two groups of three iso-genic rats housed under identical conditions was analyzed. Of 20,000gene fragments identified, only four differed in expression betweenthe groups. Each of these differences was less than fourfold in magni-tude. Thus, the false-positive rate as approximated by this experi-ment, is 1 in 5,000.

Application to rat cardiac hypertrophy. To validate its utility, themethod was applied to a well-characterized rat surgical model ofpressure overload (POL)-induced cardiac hypertrophy18,19. The ratsurgical POL model is attractive for expression analysis since the tis-sue reaction to POL features hypertrophy of cardiomyocytes andhyperplasia of endothelial, smooth muscle, and mesenchymal cellswith minimal infiltration by nonresident cells. While this process isinitially adaptive, there is ultimately a deterioration of contractilefunction accompanied by interstitial and perivascular fibrosis20,21.Two weeks after surgery there is >50% increase in the ventricularweight/body weight ratio (4.19 ± 0.52 mg/g, n = 3), compared to thesham surgery group (2.59 ± 0.52 mg/g, n = 3).

Gene expression profiling was performed on pressure-over-loaded male rat heart cDNA, sham-operated heart cDNA, and non-operated heart cDNA (n = 3 per group). A total of 18,000 genefragments per sample group were generated from 80 reactions(each with a different pair of restriction enzymes) and examinedfor expression levels. Database look-up and gene confirmationsuggested an approximately 1.5-fold redundancy in gene coverage.

Using a 1.5-fold cut-off, comparison between the POL hearts andthe sham surgery hearts yielded 74 differences (0.5%), while thepressure overload versus nonoperated hearts gave 117 differences(0.9%). When these two analyses were combined, the intersectionof fragments showing altered expression in both analyses wasmuch smaller, with only 35 fragments (0.2%) corresponding to 23differentially expressed genes (Table 1). The identity of each of thedifferentially expressed gene fragments was confirmed by oligonu-cleotide poisoning.

Our method identified genes previously known to be modulatedin cardiac hypertrophy and others that had not been previouslyassociated with this response. Among the genes previously knownto be altered were ANP, a-skeletal actin, and myosin isoformswitching22–25, in addition to the matrix proteins fibronectin26 andcollagens I and IV27. To confirm the magnitude of expressionchanges, probes from five genes were hybridized to northern blots ofRNA prepared from an independent set of six POL and sham hearts(Table 1). The results confirmed differential expression of each gene,and estimates of the quantitative changes by the two methods werein general agreement.

Distinct patterns of tissue gene expression. We identified 15known genes that had not been previously associated with modelsof cardiac hypertrophy (Table 1). Eight of these encoded extracel-lular matrix proteins, suggesting a role in the tissue response toPOL. These include several collagens, fibronectin, biglycan, p85,laminin-g1, and fibrillin, all of which are myocardial matrix pro-teins28–31, while SPARC is a multifunctional matrix proteininvolved in morphogenesis and tissue remodeling32,33. Analysis oftissue pathology (Fig. 5A) suggested histological changes consis-


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Figure 4. (A) Dilutions of 1:5,000, 1:25,000, and 1:125,000 of plasmid DNA were added to rat liver cDNA before performing GeneCalling reactionsand compared to undoped profiles (B). Dilutions of 1:1,300, 1:13,000, 1:65,000, and 1:130,000 of human cRNAs were added to HeLa poly-A+ RNAbefore cDNA synthesis and profiles were generated. (C) Relative peak intensity plotted against RNA dope ratio.

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Figure 3. Correlation study comparing reproducibility of fluorescence intensity of peaks produced from independent reactions or samples. (A)Peak intensities plotted from a single lane of electrophoresis against another lane on a different gel using the same starting cDNA and the samechemistry protocol. Evident here is the variation due to the chemistry protocol plus electrophoresis, with a variance of ~20%. (B) Mean peakheights of three replicates of one sham-treated animal compared to the mean peak heights of three replicates of another sham-treated animal,with a variance of ~5%. (There is some saturation of very abundant transcripts in one of the samples in this enzyme pair being analyzed) (C)Comparison of diseased and normal animals; outlying points significantly deviating from the mean are gene expression differences, which, onaverage, can each be seen as 8–12 outlying points, corresponding to 0.8–1.2 nucleotides as expected (intensity was collected every 0.1 nt).

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tent with this class of genes. In POL hearts the left and right ventric-ular walls were thickened with scattered areas of subacute myofibernecrosis, hemorrhage, and repair. Some sections showed mildthickening of small arteries and arterioles, while others showedfibrinoid necrosis, presumably related to hypertensive changesinduced by surgical POL. Occasional mitoses of vascular smoothmuscle cells were observed, and there were moderate accumula-tions of myxomatous material and increased cellularity surround-ing the adventitia of arterioles (Fig. 5A).

To further correlate tissue pathology with gene expressionchanges, we performed in situ hybridization on sham surgically treat-ed and POL cardiac tissue with four of the differentially expressedmatrix genes and preproenkephalin. Biglycan, a small proteoglycanthat binds collagen I and that may play a role in collagen fibril gener-ation28, was found to be expressed in the vascular media and adventi-tia of arterioles and the walls of smaller vessels in the cardiac intersti-tium (Fig. 5B, panels 1–3), the endocardial surfaces, and the valves incontrol and banded hearts (not shown). A diffuse increase in theinterstitial signal and more prominent focal accumulations of bigly-can mRNA in areas of interstitial fibrosis occurred in POL hearts. p85mRNA (a matrix component of undefined function)29 was diffuselydistributed at low levels through the parenchyma, with more intensesignal in adventitial and endocardial cells and valvular connective tis-sue in sham-surgery hearts. Increased hybridization signal was pre-sent in the banded hearts, particularly in areas of focal interstitialfibrosis (Fig. 5C, panels 3 and 6). Expression of the microfibril pro-tein fibrillin, which is mutated in Marfan syndrome31 was found infibroblast-like cells throughout the atria and ventricles of control ani-mals. In POL hearts, expression was enhanced at sites of myocardialinjury and around the walls of arteries (Fig. 5C, panel 5). Increasedexpression was also found in subendothelial cells lining the wall ofthe left atrium in POL hearts (not shown). Unlike structural compo-nents of matrix, SPARC regulates cell–extracellular matrix interac-tions without contributing directly to matrix structure32,33. Strong

Table 1. Differential gene expression in POL hypertrophya.

Gene Fragments Fold p Value Northerndifference difference

Antizyme inhibitor 1 2.0 0.08a-Skeletal actin 6 4.5 <0.001 3.7Atrial natriuretic 2 11 <0.001 17.0

peptide (ANP)a-Cardiac MHC 1 -1.9 0.02b-Cardiac MHC 1 3.6 <0.001Biglycan 7 5.2 <0.001 2.4Collagen III 1 3.8 0.06Collagen IV 1 1.8 <0.001Cytochrome 1 7.3 <0.001

oxidase ICytochrome 1 3.0 0.08

oxidase IICyclin G 1 3.3 0.0913 kDa Differentiation 1 3.6 <0.001

assoc. ProteinFibrillin 1 2.0 <0.001Fibronectin 1 3.6 0.06Gelsolin 1 4.3 <0.001Laminin g-1 1 3.8 0.07 1.4P85 1 3.5 <0.001 1.6Preproenkephalin 1 3.4 <0.001Protein kinase C- 1 -5.3 <0.001

binding protein b15Short-chain alco- 1 -1.5 <0.001

hol dehydrogenaseSPARC 1 2.6 0.07Transglutaminase 1 2.0 <0.001Zyxin 1 20.8 <0.001

aThe names of the differentially expressed genes (Column 1) are given with thenumber of fragments that were identified (Column 2), the fold modulation of thegene (Column 3), the significance of the difference (Column 4), and the expres-sion difference as detected by Northern analysis (where determined, Column 5).

Figure 5. (A) Hematoxylin and eosin staining of coronary artery branch from asham-treated (panel 1) or POL rat heart (panel 2). The POL artery showssmooth muscle hyperplasia and hypercellularity of the periadventitial tissues(arrow). A mitotic smooth muscle muscle cell is seen within the wall of theartery (arrowhead). (B) Bright-field images of biglycan (panels 1–3) and SPARC(panels 4–6) in situ hybridization. Sham-treated hearts are represented in pan-

els 1 and 4, and POL hearts in panels 2, 3, 5 and 6. Biglycan expression was also observed in spindle-shaped fibroblast-like cells in sitesof myocardial fibrosis in banded hearts (panel 3). SPARC was expressed by endothelial cells (panel 5, arrow) as well as spindle-shapedcells lining the adventitia of arteries (arrowhead), and showed increased expression in POL arteries (panel 6). Like biglycan, SPARC washighly expressed in fibroblast-like cells at sites of myocardial fibrosis (panel 6, arrowhead). (C) In situ hybridization of preproenkephalin(panels 1 and 4), fibrillin (panels 2 and 5), and p85 (panels 3 and 6) in sham surgery (panels 1 and 2) and POL myocardium (panels 3, 4, 5,and 6). Panels 1 and 4 show expression of preproenkephalin by myocytes (arrowhead), with increased expression in POL hearts. Panels 2and 5 show expression of fibrillin (arrowhead). In normal hearts fibrillin was expressed in spindle-shaped fibroblast-like cells in the adven-titia of arteries and in similar cells throughout the myocardium (panel 2). In POL hearts fibrillin was additionally expressed at sites ofmyocardial fibrosis (panel 5). Panels 3 (bright field) and 6 (dark field) are the same POL field showing expression of p85 in spindle-shapedcells, particularly in areas of focal interstitial fibrosis (arrowhead).

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expression of SPARC mRNA in endothelial cells and pericytes wasseen throughout the hearts of sham-treated controls. In POL hearts,expression increased at sites of myocardial damage, around arteries(Fig. 5B, panels 5 and 6), and in cells lining the wall of the left atrium(not shown).

Preproenkephalin has been shown to be expressed by myocytesand increased in response to myocardial infarction34,35. In bothsham-treated and POL tissues preproenkephalin mRNA was consis-tently greater in the left than in the right ventricle by in situhybridization (not shown). No expression was observed in atria. Insham-treated hearts, expression was largely confined to a subset ofcardiac myocytes on the inner third of the left and right ventricles. InPOL hearts the same pattern was observed, but the intensity of signalwas increased (Fig. 5C, panel 4). Additionally, spindle-shaped cells,expressing high levels of preproenkephalin, were seen in and aroundareas of myocardial fibrosis.

We have identified a small proportion of differentially expressedgenes (0.2%) during cardiac hypertrophy. This is remarkable given a60% increase in ventricular mass. One interpretation of this result isthat cardiac tissue responds to increased hemodynamic load primarilyby proportional growth of the myocardium36,37. Some of the genesidentified may, in fact, represent changes in gene expression associatedwith heart failure rather than hypertrophy. In support of this are find-ings from a hypertensive rat model of hypertrophy38. In this model,compensatory hypertrophy could be distinguished from hypertrophywith heart failure by the elevated expression of genes involved in extra-cellular matrix production. The identification of a variety of knownand new matrix-related genes suggests that the rat POL model at twoweeks may have already initiated disproportionate fibrotic remodelingthat will predispose to failure.

Our method has advantages and disadvantages with respect toother expression profiling methods. It does not provide an absoluteabundance for an RNA of interest, nor is it well suited to comparevery different tissue types. However, the reproducibility of the datacan permit development of cumulative databases of tissue and cellexpression profiles. The method is not subject to abundance-depen-dent redundancy in sampling, and provides a survey of expressionlevels for most genes present at a level of ³1:100,000. GeneCallingalso provides a relatively rapid and comprehensive system for thediscovery of new, differentially expressed genes.

Experimental protocolRNA isolation. Total RNA was isolated with Trizol (Life Technologies, GrandIsland, NY) using 0.1 volume of bromochloropropane for phase separation(Molecular Research Center, Cincinnati, OH), and treated with DNase I(Promega, Madison, WI) in the presence of 0.01 M dithiothreitol (DTT) and1 U/ml RNasin (Promega). Following phenol/chloroform extraction, RNAquality was evaluated by spectrophotometry and formaldehyde agarose gelelectrophoresis, and yield was estimated by fluorometry with OliGreen(Molecular Probes, Eugene, OR). Poly-A+ RNA was prepared from 100 mgtotal RNA using oligo(dT) magnetic beads (PerSeptive, Cambridge, MA),and quantified with fluorometry.

First-strand cDNA was prepared from 1.0 mg of poly(A)+ RNA with 200pmol oligo(dT)25V (V = A, C or G) using 400 U of Superscript II reverse tran-scriptase (BRL). Second-strand synthesis was performed at 16°C for 2 h afteraddition of 10 U of E. coli DNA ligase, 40 U of E. coli DNA polymerase, and 3.5U of E. coli RNase H (all from BRL). T4 DNA polymerase (5 U) was added,incubated for 5 min at 16°C, followed by treatment with arctic shrimp alkalinephosphatase (5 U; United States Biochemicals, Cleveland, OH) at 37ºC for 30min. cDNA was purified by phenol/chloroform extraction, and the yield wasestimated using fluorometry with PicoGreen (Molecular Probes).

cDNA fragmentation was achieved by digestion in a 50 ml reaction mixturecontaining 5 U of restriction enzyme and 1 ng of double-stranded cDNA. Weperformed 80 separate sets of cDNA fragmentation reactions, each with a dif-ferent pair of restriction enzymes. These were then ligated to complementaryamplification tags with ends compatible to the 5¢ and 3¢ ends of the fragmentsat 16°C for 1 h in 10 mM ATP, 2.5% PEG, 10 units T4 DNA ligase, and 1´ lig-

ase buffer. Amplification was then performed after addition of 2 ml 10 mMdNTP, 5 ml 10´ TB buffer (500 mM Tris, 160 mM (NH4)2SO4, 20 mM MgCl2,pH 9.15), 0.25 ml Klentaq (Clontech Laboratories, Palo Alto, CA): PFU(Stratagene, La Jolla, CA) (16:1), 32.75 ml H2O. Amplification was carried outfor 20 cycles (30 s at 96°C, 1 min at 57°C, 2 min at 72°C), followed by 10 minat 72°C. PCR products were purified using streptavidin beads (CPG, LincolnPark, NJ). After washing the beads twice with buffer 1 (3 M NaCl, 10 mMTris-HCl, 1 mM EDTA, pH 7.5), 20 ml of buffer 1 was mixed with the PCRproduct for 10 min at room temperature, separated with a magnet, andwashed once with buffer 2 (10 mM Tris, 1 mM EDTA, pH 8.0). The beadswere then dried and resuspended in 3 ml of buffer 3 (80% (vol/vol) for-mamide, 4 mM EDTA, 5% TAMRA- or ROX-tagged molecular size standards(PE-Applied Biosystems, Foster City, CA). Following denaturation (96°C for3 min), samples were loaded onto 5% polyacrylamide, 6 M urea, 0.5 ´ TrisBorate EDTA ultrathin gels and electrophoresed on a Niagara instrument.PCR products are visualized by virtue of the fluorescent FAM label at the 5¢end of one of the PCR primers, which ensures that all detected fragmentshave been digested by both enzymes.

The primary components of the Niagara gel electrophoresis system are aninterchangeable horizontal ultrathin gel cassette mounted in a platformemploying stationary laser excitation and a multicolor CCD imaging system.Each gel cassette is loaded in four cycles of 12 wide (48 lanes total) directlyfrom a 96-well plate using a robotic arm. The Niagara system has the advan-tage of high throughput, with separation of fragments between 30 and 450bases in 45 min.

Gel interpretation. Electrophoresis data was processed using the OpenGenome Initiative (OGI) software. Gel images were first visually checked andtracked. Each lane contains the FAM-labeled products of a single reaction plusa sizing ladder spanning the range from 50 to 500 bp. The ladder peaks pro-vide a correlation between camera frames (collected at 1 Hz) and DNA frag-ment size in base pairs. After tracking, lanes are extracted and the peaks in thesizing ladder are found. Linear interpolation between the ladder peaks con-verts the fluorescence traces from frames to base pairs. A final quality con-trol step checks for low signal-to-noise, poor peak resolution, missing lad-der peaks, and lane-to-lane bleed. Data that pass all of these criteria aresubmitted as point-by-point length versus amplitude addresses to anOracle 8 database.

Difference identification. For each restriction enzyme pair in each sam-ple set a composite trace was calculated, compiling all the individual samplereplicates followed by application of a scaling algorithm for best fit to nor-malize the traces of the experimental set versus that of the control. Thescaled traces are then compared on a point-by-point basis to define areas ofamplitude difference that meet the minimum prespecified threshold for asignificant difference. Once a region of difference has been identified, thelocal maximum for the corresponding traces of each set is then determined.The variance of the difference is calculated by the following expression:

s2D(j) = l1(j)2s2

Total(j:S1) + l2(j)2s2Total(j:S2)

where ll(j) and l2(j) represent scaling factors and (j:S) represents the tracecomposite values over multiple samples. The probability that the differenceis statistically significant is calculated by

P(j)=1–º-D

D

dy (1/Ã{2¹sD2}) exp (-y2/2s2

D)

where y is the relative intensity. All difference peaks are stored as uniquedatabase addresses in the specified expression difference analysis.

Gene confirmation by oligonucleotide poisoning. Restriction fragmentsthat map in end sequence and length to known rat genes are used as tem-plates for the design of unlabeled oligonucleotide primers. An unlabeledoligonucleotide designed against one end of the restriction fragment is addedin excess to the original reaction, and is reamplified for an additional 15cycles. This reaction is then electrophoresed and compared to a control reac-tion reamplified without the unlabeled oligonucleotide to evaluate the selec-tive diminution of the peak of interest.

RNA doping. DNA templates for RNA in vitro transcription were generat-ed by PCR amplification using cloned human cDNAs as templates. PCRprimers were complementary to plasmid sequences flanking the cDNAinserts. In addition, the sense primer contained the T7 RNA polymerase con-sensus sequence, and the antisense primer included a stretch of 25thymidines for the generation of polyadenylated transcripts. In vitro tran-scription was performed using the MaxiScript transcription kit (Ambion,


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Austin, TX). The transcripts were poly-A selected on biotin-oligo(dT)25

bound to streptavidin MPG beads (CPG Inc.). The RNA products ranged insize between 1,100 and 2,000 nt. The integrity of the products was monitoredby agarose gel electrophoresis and the concentration determined by fluorom-etry using RiboGreen dye (Molecular Probes) on a SpectraFluor fluorometer(Tecan, Grundig, Austria). The in vitro transcribed RNAs were mixed atdefined ratios with HeLa cell poly-A+ RNA (American Type CultureCollection, Manassas, VA) and the RNA was converted to cDNA and subject-ed to GeneCalling chemistry and analysis as described.

POL hypertrophy. Male Sprague-Dawley (SD) rats (Charles RiverBreeding Laboratories, Wilmington, MA) aged six to seven weeks were accli-mated to the facility for at least one week before surgery, fed pelleted rat chowand water ad libitum, and housed in a room with controlled light and tem-perature. The experimental procedures, which were approved by Genentech’sInstitutional Animal Care and Use Committee, conform to the guiding prin-ciples of the American Physiological Society. The induction of pressure over-load by partial ligation of the abdominal aorta in rats was as described39,40. Attwo weeks after surgery, animals were anesthetized, and hearts were rapidlyremoved by thoracotomy, blotted of excess blood before dissection andweighing, then frozen with liquid nitrogen.

Northern analysis. 1 mg of poly(A)+ RNA from four to five sham-treated andPOL ventricles was electrophoresed in agarose/formaldehyde gels, blotted tonylon filters, and probed with fill-in oligonucleotides labeled with 33P-dCTP.The hybridization was quantitated with a storage phosphor imaging plate usingthe Fuji BAS2000 (Fuji Photo Film Co., Tokyo, Japan). Blots were stripped andreprobed for GAPDH to normalize gene expression levels. Induction levels areexpressed as the ratio of average normalized hybridization signals.

Histology and in situ hybridization. Tissues were formalin fixed and paraffinembedded, 10 mm thick sections were cut, stained with hematoxylin and eosin(H&E) for histology, or used for in situ hybridization. 32P-labeled sense and anti-sense riboprobes were obtained by T3 (antisense) or T7 (sense) RNA polymerasetranscription of PCR fragments from each cDNA. Specific transcript sizes (innucleotides) were 781 (preproenkephalin), 485 (fibrillin), 665 (SPARC), 491(biglycan), and 595 (p85). For hybridization, sections were deparaffinized anddigested with 20 mg/ml of proteinase K at 37°C for 15 min, before applying theprobe. After overnight hybridization, sections were treated with 20 mg/ml RNaseA, followed by a high-stringency wash in 0.1 ´ saline sodium citrate at 55°C.Sections were dehydrated, dipped in NTB-2 emulsion, and exposed for one tofour weeks. Control hybridizations with sense probes gave negligible signals.

A technical description of GeneCalling can also be found in US Patent5,871,697.

AcknowledgmentsThe authors thank Dr. Richard Lifton for helpful comments on this manuscript,the Genentech in situ hybridization laboratories for technical assistance withanatomical pathology and Dr. Anne Ryan for interpretation of histopathology.

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