Scalable Transcriptional AnalysisRoutine—Multiplexed Quantitative Real-TimePolymerase Chain Reaction Platform for GeneExpression Analysis and Molecular Diagnostics
Elizabeth P. Garcia,*† Lori A. Dowding,*Lawrence W. Stanton,‡ and Vladimir I. Slepnev*†
From Primera BioSystems,† Providence, Rhode Island; Sention,*
Providence, Rhode Island; and Genome Institute of Singapore,‡
Singapore, Singapore
We report the development of a new technology forsimultaneous quantitative detection of multiple tar-gets in a single sample. Scalable transcriptional anal-ysis routine (STAR) represents a novel integration ofreverse transcriptase-polymerase chain reaction andcapillary electrophoresis that allows detection of doz-ens of gene transcripts in a multiplexed format usingamplicon size as an identifier for each target. STARdemonstrated similar or better sensitivity and preci-sion compared to two commonly used methods,SYBR Green-based and TaqMan probe-based real-timereverse transcriptase-polymerase chain reaction.STAR can be used as a flexible platform for building avariety of applications to monitor gene expression,from single gene assays to assays analyzing the ex-pression level of multiple genes. Using severe acuterespiratory syndrome (SARS) corona virus as a modelsystem, STAR technology detected single copies of theviral genome in a two-gene multiplex. Blinded studiesusing RNA extracted from various tissues of a SARS-infected individual showed that STAR correctly iden-tified all samples containing SARS virus and yieldednegative results for non-SARS control samples. Usingalternate priming strategies, STAR technology can beadapted to transcriptional profiling studies withoutrequiring a priori sequence information. Thus, STARtechnology offers a flexible platform for developmentof highly multiplexed assays in gene expression anal-ysis and molecular diagnostics. (J Mol Diagn 2005,7:444–454)
Nucleic acid testing of clinical samples and tissues isincreasingly widespread and applied to various areas ofmedicine and diagnostics including pathogen identifica-tion,1 blood bank testing,2 cancer recurrence,3 and pre-diction of clinical outcome.4 The competitive landscapeof available technologies is characterized by clear sep-
aration of very sensitive and quantitative methods mea-suring single bioanalytes [real-time polymerase chain re-action (PCR), transcription mediated amplification, ligasechain reaction, rolling circle amplification, and so forth]and methods capable of multiplexing thousands of genes(DNA microarrays, serial analysis of gene expression,differential display, and so forth) with less sensitivity andquantitative ability compared to single gene methods.The recent introduction of technologies capable of ana-lyzing a number of analytes or biomarkers in a high-throughput multiplex configuration in clinical diagnos-tics5,6 and gene expression analysis (HT Genomics,www.htgenomics.com) demonstrates a growing trend to-ward development of multiplex assays. The future of nu-cleic acid testing requires better multiplexing abilities thatmaintain or exceed the current levels of sensitivity.
Although real-time PCR is the most common methodusing optical detection, others such as transcription-me-diated amplification, ligase chain reaction, and stranddisplacement amplification are also widely used andmarketed for nucleic acid testing diagnostics.7,8 Many ofthese techniques depend on the detection and quantifi-cation of fluorescent molecules whose signal increases inproportion to the amount of amplified nucleic acid gen-erated. Yet multiplexing capabilities for these methodsare limited due to the overlapping absorption and emis-sion spectra of available fluorophores thus restricting thenumber of multiplexed targets to four or five.9,10
At the opposite extreme, several methods have beendeveloped that have excellent multiplexing capabilitiesallowing the analysis of large amounts of genomic infor-mation. The most widely used method depends on DNAmicroarrays, a technique that detects expression of up tothousands of genes based on optical detection of hybrid-ized fluorescently labeled DNA probes combined withspatial positioning.11–13 Serial analysis of gene expres-sion14 and differential display15 also allow the transcrip-tome of two samples to be compared with the addedadvantage that a priori sequence knowledge is not re-quired. Despite this, each of these techniques has sev-
Supported by Sention, Inc.
Accepted for publication May 3, 2005.
Address reprint requests to Vladimir Slepnev, 4 Richmond Sq., Provi-dence, RI 02906. E-mail: [email protected].
Journal of Molecular Diagnostics, Vol. 7, No. 4, October 2005
eral drawbacks. They lack sensitivity, show poor quanti-fication capabilities, and require large amounts ofbiological material.16 Finally, each is a long multistepprocess that requires a high level of technical expertisethat can contribute to variability in data obtained.
We have developed STAR (scalable transcription anal-ysis routine), a gene expression analysis platform thatrepresents an innovative integration of real-time multiplexPCR and capillary electrophoresis (CE), allowing the si-multaneous quantitative measurement of multiple targetsin a single sample with high sensitivity. Specificity of PCRamplification is due to appropriate primer choice andreaction conditions. Because CE allows accurate sizedetermination of fluorescently labeled nucleic acids from50 to 1000 bases with single base precision, assays canbe conducted simultaneously for dozens of targetswhose identities are defined by the specific size of itscorresponding amplicons, while maintaining quantifica-tion capabilities equal to or better than those observedwith established real-time PCR methods. Although initialproof-of-concept experiments were performed manually,in the course of this work, we automated aliquot dispens-ing during PCR amplification, and are currently workingon integration of a fully automated system that will inte-grate aliquot dispensing with CE separation. STAR is fast,cost-effective, and has a large dynamic range. Here wepresent STAR technology and its application to diagnos-tics and gene expression analysis.
Materials and Methods
Description of STAR Technology
In a typical STAR experiment (diagrammatically shown inFigure 1A), a PCR reaction is set up in a single tubecontaining the analyte, common PCR reagents (eg, DNApolymerase, dNTPs), and, for each target to be amplified,gene-specific primers where at least one of each pair islabeled with a fluorophore. PCR primers are designed foreach target so that the amplicon length serves as aunique identifier for each particular target. Each ampliconmust vary by at least 5 nucleotides in size. Aliquots of themultiplex PCR reaction are removed after successivePCR cycles and separated by CE. Amplification curvesare reconstructed based on the area under each ampli-fied target. As for conventional real-time PCR, cyclethresholds (CT) can be determined from the graphsthereby allowing the determination of initial copy numberfor each amplified template. For applications that quantifyRNA levels, STAR can be modified to a one-step reversetranscriptase (RT)-PCR reaction (see below).
One-Step STAR Protocol
RNA template and forward and reverse gene-specificprimers were added to a mixture containing 1� Strat-agene buffer (catalog no. 600532, modified to contain0.1% Triton X-100, 1.5 mmol/L MgCl2) and 0.3 U/�l ofStrataScript RTase (Stratagene, La Jolla, CA) and reversetranscribed at 45°C for 50 minutes, followed by 2 minutes
at 94°C. At least one of each primer pair was fluores-cently labeled with FAM. The PCR protocol consisted of31 to 44 cycles of 94°C for 30 seconds, 60°C for 30seconds, and 72°C for 1 minute. While ramping up to thefirst 72°C extension, 1 U of Vent(Exo�) DNA polymerase(New England Biolabs, Beverly, MA) was added. After apredetermined number of cycles, 3 �l aliquots were col-lected at the end of successive cycles and immediatelyadded to 7 �l of formamide containing ROX-labeled DNAstandards (BioVentures, Freesboro, TN) followed by heatdenaturation and separation by CE using the 3100 Ge-netic Analyzer (Applied Biosystems, Foster City, CA).Samples were injected at 3 kV for 20 seconds and sep-arated at 15 kV on POP4 polymer. Data were analyzed forpeaks and relative areas as determined by Gene Scanversion 3.7.1 software provided with the instrument. Datacollected were analyzed to construct amplification curvesusing proprietary software, AnalySTAR, developed forSention.
RNA Isolation from Rat Brain
Rats were anesthetized with isoflurane (Henry Schein,Melville, NY) and decapitated. Brains were quickly re-moved, flushed with oxygenated artificial cerebrospinalfluid, and kept cold during the dissection procedure.Extraction of total RNA was performed in a two-stepprotocol using Trizol (Invitrogen, Carlsbad, CA) and RNAeasy kit (Qiagen, Valencia, CA) as per manufacturers’instructions.
Figure 1. Description of STAR technology. A: Diagrammatic representationof STAR technology. See text for detailed explanation. Abbreviations: GSP,gene-specific primer; CT, cycle threshold. To illustrate the process, threegenes (arc, homer1a, and zif268) were amplified from 100 ng of rat brain totalRNA in a multiplex format using 0.5 �mol/L of each gene-specific primer.Forward primers were fluorescently labeled. Aliquots collected betweencycles 12 and 35 were separated by CE and analyzed by GeneScan version3.7.1 generating electropherograms. B: Successive electropherograms fromcycles 19 through 22 are shown. Peaks representing arc, homer1a, and zif268are marked with an asterisk. Small repeating peaks represent DNA molec-ular size markers. C: Amplification curves for arc (filled triangles), homer1a(filled circles), and zif268 (filled squares) were reconstructed by plottingthe area under each peak against cycle number.
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Construction of Artificial Constructs
HA-tagged amphiphysin gene constructs17 were ampli-fied using vector-specific 5�- and 3�-primers such that theT7 promoter was incorporated upstream of each con-struct. The 3�-primer contained a 15-nucleotide d(T) tailthat allowed addition of a poly(A) during RNA prepara-tion. PCR products were gel purified and transcribedusing T7 RNA polymerase (Stratagene) generating artifi-cial transcripts that contained common 5� and 3� nucleicacid sequences at each end and a poly(A) tail at the3�end.
Primer Sequences
Primers for targets were designed using PrimerSelect(DNA Star, Madison, WI). Unlabeled primers were or-
dered from Qiagen, fluorescently labeled primers fromSynthegen (Houston, TX) or Applied Biosystems, andTaqMan probes from Applied Biosystems. Primer se-quences are shown in Table 1.
TaqMan Probe-Based and SYBR Green-BasedReal-Time RT-PCR Protocols
Each sample was serially diluted 2-fold from the startingconcentration in 0.1% bovine serum albumin and ampli-fied in a one-step RT-PCR protocol using 0.5 �mol/Lprimers. For TaqMan probe-based real-time RT-PCR as-says (TaqMan) using 0.066 �mol/L probe, cycling pa-rameters were 1 cycle of 45°C for 45 minutes, 95°C for 10seconds followed by 40 cycles of 95°C for 15 seconds,57°C for 10 seconds, 57°C for 1 minute. SYBR Green-
Table 1. Nucleic Acid Sequence of Primers Used in This Study
5�-�FAM�TGGGGCAGCCGAGAAGGAC-3�ST1 Alternative priming 5�-�FAM�CGCTCGTAGTCGAACGCCTAACCA-3�ST2 Alternative priming 5�-�FAM, VIC, or NED�CGACGTATGCGTAACCCGTATCGT-3�UT2 Alternative priming 5�-GCGGCGCCTATCTTACTATST1-dT14-VN Alternative priming 5�-CGCTCGTAGTCGAACGCCTAACCATTTTTTTTTTTTTTVN-3�ST2-dT14-VN Alternative priming 5�-CGACGTATGCGTAACCCGTATCGTTTTTTTTTTTTTTTVN-3�UT2-HA Alternative priming 5�-GCGGCGCCTATCTTACTATCCAGACTA-3�Rep1B SARS multiplex 5�-AAGCCTCCCATTAGTTTTCCATTA-3�
*For STAR experiments, the first oligo listed is FAM labeled; for SYBR and TaqMan, oligos are unlabeled.FAM, 6-carboxyfluorescein; TAMRA, 6-carboxytetramethylrhodamine.
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based real-time RT-PCR (SYBR) cycling parameters were1 cycle of 45°C for 30 minutes, 95°C for 7 minutes fol-lowed by 40 cycles of: 95°C for 30 seconds, 57°C for 30seconds, 72°C for 1 minute. For both assays, the anneal-ing temperature was reduced to 52°C for zif268 amplifi-cation. STAR cycling parameters were identical to that ofSYBR except that RT was performed at 45°C for 50minutes followed by 95°C for 2 minutes before PCR am-plification. The annealing temperature for the multiplexSTAR reaction was 55°C.
STAR Protocol Using Alternative PrimingStrategies
For reverse transcription (RT), RNA template and se-quence-tagged reverse primers (5 �mol/L) were addedto 58% glycerol, heated at 70°C for 10 minutes, then puton ice for 2 minutes. Buffer (final concentrations: 50mmol/L Tris-HCl, pH 8.3, 75 mmol/L KCl, 3 mmol/LMgCl2, 0.01 mol/L dithiothreitol, 0.8 mmol/L dNTP, 0.2mg/ml bovine serum albumin, 20% trehalose), 3.2 U/�l ofSuperscript II RNase H� reverse transcriptase (SSRTII,Invitrogen) and 1 U/�l of RNAsin (Ambion, Austin, TX)were added and reverse transcribed at 45°C for 20 min-utes, followed by denaturation at 75°C. A second roundof RT at 48°C for 20 minutes was initiated with the addi-tion of 50 U SSRTII followed by a third round of RT at 52°Cfor 20 minutes after a 2-minute 80°C denaturation step.Samples were alkaline treated with 0.04 mol/L NaOH(final concentration) for 15 minutes at 65°C, followed byaddition of Tris, pH 7.5, to a final concentration of 70mmol/L. Resultant cDNAs were purified using the QIA-quick gel extraction kit (Qiagen) as per the manufactur-er’s instructions, except that 360 �l of QG buffer wasadded to each sample. For second strand synthesis,purified cDNA in 40 mmol/L Tris (pH 7.5), 20 mmol/LMgCl2, 50 mmol/L NaCl, 0.2 mmol/L dNTPs was heatdenatured at 95°C for 1 minute followed by addition of 1.6�mol/L second strand primers and continued denatur-ation at 95°C for 4 minutes. The reaction was ramped to37°C, 0.5 U/�l Sequenase DNA polymerase (USB, Cleve-land, OH) was added and incubated for 1 hour. DNA waspurified using the QIAquick gel extraction kit as above.Primers (0.5 �mol/L) were added and PCR amplificationproceeded in 10 mmol/L KCl, 10 mmol/L (NH4)2SO4, 20mmol/L Tris-HCl (pH 8.8), 2 mmol/L MgSO4, 0.1% TritonX-100, 0.2 mmol/L dNTPs, 20% Q solution (Stratagene),2% dimethyl sulfoxide, 2 U Vent DNA polymerase over-laid with mineral oil. PCR protocol: 95°C for 5 minutesfollowed by a variable number of cycles of 95°C for 30seconds, 56°C for 30 seconds, 72°C for 1 minute.Three-�l aliquots were collected at the end of each cyclefor 24 successive cycles and processed as described.
SARS Samples
All RNA samples derived from SARS corona virus (SARS-CoV)-infected Vero cells,18 blinded clinical samples Athrough H, and normal human tissues were obtained fromthe Genome Institute of Singapore. RNA extractions were
performed using High Pure Viral RNA kit (Roche Diag-nostics, Mannheim, Germany). The copy number ofSARS-CoV RNA was quantified against reference stan-dards using the LightCycler SARS-CoV quantification kit(Roche Diagnostics).
SARS Amplification by One-Step STAR
SARS-CoV RNA samples were diluted in Escherichia colitRNA at 20 �g/ml. One-step amplification of SARS tran-scripts was performed as described above using appro-priate fluorescently labeled forward and reverse primersfor 1 cycle of 45°C for 50 minutes, 94°C for 2 minutes,followed by 44 cycles of 95°C for 30 seconds, 60°C for 30seconds, 72°C for 1 minute.
Results
Proof of Principle: STAR Technology Can BeUsed to Multiplex RT-PCR Transcripts from aComplex Background Generating Real-TimeAmplification Curves
To illustrate the STAR process, a one-step multiplex am-plification of three transcripts, arc,19 homer1a,20 andzif26821 was performed from 100 ng of total rat brain RNAand analyzed. The upper primer of each primer pair wasfluorescently labeled with FAM. Three-�l aliquots werecollected after successive PCR cycles 12 through 35,separated by CE and analyzed. Peaks representing eachof the amplified targets are depicted in sequential elec-tropherograms derived from PCR cycles 19 through 22(Figure 1B). Note the growing peaks with consecutivecycles and also the lack of nonspecific amplification. Thearea under the peaks corresponding to each target wasplotted against cycle number to generate the amplifica-tion curves shown in Figure 1C. As shown, STAR tech-nology clearly detected the presence of all three tran-scripts in a complex background generating typicalRT-PCR amplification curves.
Comparison of STAR Technology to TaqManProbed-Based and SYBR Green-Based Real-Time RT-PCR
STAR technology is a further extension of multiplex real-time PCR that allows a much greater degree of multiplex-ing compared to existing technologies. STAR benefitsfrom the integration of PCR and CE in which specificity ofPCR amplification is due to appropriate primer choiceand reaction conditions, and CE allows resolution of DNAfragments that differ by a few bases. The multiplexingpower of STAR is a result of using amplicon size as aunique identifier for particular genes or biomarkers in-stead of fluorescent color or melting temperature used inother methods. In STAR experiments, a multiplex PCRreaction is sampled after each PCR cycle and subse-quently separated by CE. The area under peaks corre-
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sponding to specific PCR products are quantified andplotted as a function of cycle number to generate ampli-fication plots, which can be analyzed using current algo-rithms of real-time PCR analysis (Figure 1; see Materialsand Methods for a complete description). Currently, Taq-Man probe-based (TaqMan) and SYBR Green-based(SYBR) real-time RT-PCR are the methods of choice fordetection and quantification of transcripts. We compareddetection of three genes (arc, homer1a, and zif268) in acomplex background using TaqMan, SYBR, and STAR,individually or as a three-gene multiplex. Twofold seriallydiluted samples of total brain RNA were used as sampletemplates. To allow direct comparison between proto-cols, the PCR primer sequences used for each assaywere identical with the exceptions that TaqMan hydroly-sis probe was added for TaqMan assays and the forwardPCR primers were fluorescently labeled for STAR assays.Amplification efficiencies and amplification curves ob-served for STAR are similar to that of TaqMan and SYBRfor all three genes examined, but with higher sensitivity(Figure 2, Table 2). Cycle thresholds are six and fourcycles sooner for STAR protocols than for TaqMan orSYBR protocols, respectively, representing a 64-fold in-crease in STAR detection limits as compared to TaqManor SYBR.
Amplification Efficiency Is Constant between SingleGene and Multigene Amplifications
One concern in multiplex analysis of transcripts byreal-time RT-PCR is that the amplification efficiencies ofgiven transcripts may be influenced by the presence ofmultiple transcripts undergoing amplification. Compari-son of the amplification efficiencies observed for arc,homer1a, or zif268 show that they are not significantly
different whether performed in multiplex or as individualreactions (Table 2). We do, however, find that the cyclethreshold is often lower in multiplexed samples than sam-ples amplified individually. This is likely due to moreefficient or promiscuous priming during reverse tran-scription resulting from the increased number of primerspresent in multiplex reactions that can anneal to RNAtemplates at low reaction temperatures (42°C; Figure 2).
Sensitivity and Reproducibility of STAR Assays
To address the absolute sensitivity of STAR assays, weperformed experiments using artificial transcripts spikedinto carrier tRNA (20 �g/ml; Sigma, St. Louis, MO). Threeartificial transcripts [VS31 (450 bases), VS32 (377 bases),and VS85 (544 bases)] were 10-fold serially diluted from3,000,000 to 3 copies and PCR amplified in multiplexusing a common pair of fluorescently labeled primers aseach transcript contains common sequences at their 5�and 3� ends (see Materials and Methods). STAR detected3 copies of each of the multiplexed transcripts and lin-early detected samples from 3,000,000 to 30 copies forall transcripts with efficiencies ranging from 84.5 to95.3%. Amplicons were not detected in negativecontrols.
To determine the precision of multiplex STAR assayswithin runs, we performed four identical reactions thatcontained 300 copies of each artificial transcript dilutedin carrier RNA (Figure 3B). Cycle thresholds were 25.9 �0.48, 23.8 � 0.44, and 26.8 � 0.50 for VS31, VS32, andVS85, respectively, throughout the four runs demonstrat-ing reliable reproducibility within STAR assays. Amplifi-cation curves are shown for VS32 (Figure 3B). The meanamplification efficiency was 1.82 � 0.06.
Figure 2. STAR technology is comparable to TaqMan probe-based and SYBR Green-based real-time RT-PCR. Detection of endogenous levels of arc (a), homer1a(b), and zif268 (c) in rat brain total RNA were assessed by three real-time PCR methods: SYBR (circles), TaqMan (triangles), and STAR either as an individualreaction (squares) or as part of a multiplex reaction (diamonds). CTs were determined from PCR amplifications performed from twofold serially diluted totalrat brain RNA (400 to 0.78 ng) and plotted. For SYBR and TaqMan, CTs were calculated using Bio-Rad ICycler software (Hercules, CA).
Table 2. Comparison of Three Real-Time Methods for Gene Expression Analysis
Multiple Transcripts Covering a Wide Range ofExpression Levels Can Be Amplified andQuantified Simultaneously in a Single TubeUsing STAR Technology
To illustrate the multiplexing capacity of STAR, we per-formed simultaneous amplification of 12 transcripts froma sample of total rat brain RNA using a one-step STARprotocol (Figure 4). Targets were selected so that thepredicted amplicon size was unique to each transcript.Cycle thresholds calculated for the various transcriptsranged from 1.3 for 18S rRNA to 23.7 for Nell-2 with meanamplification efficiencies of 73–92% demonstrating thateven in the case in which the expression levels of 12target genes varied over several orders of magnitude,transcripts were detected and amplified exponentially.
Gene Profiling Using Alternate PrimingStrategies
Sample-to-Sample Comparisons
The STAR platform can be modified so that the geneexpression patterns of two or more samples can be as-sessed in a single real-time PCR reaction if sequencetags22 are incorporated into each set of primers. In thecase of RNA analysis, each sample is separately reversetranscribed using composite primers consisting of a se-quence tag (ST1 or ST2) fused to gene-specific se-quences. The sequence tags act as identifiers for thesample source. Resultant cDNAs are pooled and PCRamplified using a simplified primer set: reverse primersST1 and ST2, each labeled with a different fluorophorethereby allowing the sample source to be identified byspecific color, and a gene-specific primer for each targeteffectively reducing the number of required PCR primersin half (n sequences require n � 2 PCR primers). Thisapproach dramatically decreases the complexity of PCRamplification thus allowing multiplexing even greaternumbers of genes/biomarkers. This approach was usedto perform comparative real-time quantification of threetranscripts in drug-treated versus saline-treated tissuesamples normalized by comparing expression levels of18S rRNA. Results obtained using STAR technology con-firmed the expression levels determined using microar-ray- and SYBR Green-based real-time RT-PCR (data notshown due to space limitation).
Transcriptional Profiling
With a few modifications the priming strategies de-scribed above can be adapted to transcriptional profilingof two samples even for unknown genes (Figure 5A).
Figure 3. STAR is sensitive and reproducible. A: Artificial transcripts were10-fold serially diluted from 3,000,000 to 3 copies in E. coli tRNA (20 �g/ml)and amplified as a three-gene multiplex STAR assay using (FAM)L-HA and(FAM)pcDNA3L primers. CT versus copy number plots are shown for VS31(diamonds), VS32 (squares), and VS85 (triangles). Insufficient data pointswere obtained for VS31 and VS85 to allow CT calculation at three copies. R2
values were greater than 0.99. Amplification efficiency � 10(�1/slope) � 1. B:To demonstrate reproducibility, four samples, each containing 300 copies ofartificial transcripts VS31, VS32, and VS85 in a background of E. coli tRNA (20�g/ml), were multiplex amplified as above. Amplification curves for VS32are represented by open circles, squares, triangles, and diamonds.
Figure 4. Multiplex amplification of 12 endogenous genes by STAR technology.Twelve endogenous genes were multiplex amplified using gene-specific primersfrom 400 ng of total rat brain RNA in a one-step STAR protocol. One of eachprimer pair was fluorescently labeled. Aliquots from cycles 2 to 33 were analyzedby CE to generate amplification curves shown. Abbreviations and symbols: 18S,dark blue closed squares; actin, purple closed squares; BMP, orange filledcircles; inhibin, black closed squares; PKC, green closed circles; LDH, redclosed triangles, DGK, purple open squares; EGR3, orange open circles;arc, blue open squares; PIPK, green open circles; Nell2, orange opentriangles; NSE, black open squares.
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Samples are separately reverse transcribed using prim-ers that contain a sample-specific sequence tag (ST1 orST2) fused to poly(T)n-VN that anchors the primer to the5�-end of the poly(A) tail. Samples are then mixed fol-lowed by second strand synthesis reaction using forwardprimers that combine a second sequence tag (UT2)fused to a short arbitrary nucleotide sequence (hexameror octamer) that can prime at many loci. Based on ran-dom occurrence, hexamers should occur every 4096bases and octamers every 65,536 bases. However, the
frequency of hexamers in human expressed genome isnot random and shows considerable bias (V. Cheung,personal communication). By selecting arbitrary se-quences with care, a series of second strand primers canbe developed that result in amplification of hundreds tothousands of cDNAs per reaction based on the currentexpressed sequence tag database. We estimate that95% of the expressed genome can be covered with 20second strand primers coupled to hexamers. Samplesare then PCR amplified using reverse primers FAM-
Figure 5. Alternative priming strategy using two sequence tags. A: Schematic representation depicting the incorporation of two sequence tags during RT-PCR.Incorporation of sample-specific sequence tags during RT is followed by incorporation of a second sequence tag during second strand synthesis. PCR thenproceeds using three primers, each of the sample-specific reverse sequence tags fluorescently labeled, and the common forward sequence tag. Aliquots arecollected, separated, and analyzed by CE followed by analysis of relevant fragments. B: Total rat brain RNA samples (1.75 �g) spiked with 100 pg of VS31 (top)or unspiked (bottom) were separately reverse transcribed using sample-specific sequence tags (ST1-dT14-VN and ST2-dT14-VN) followed by RNA hydrolysis,purification, and sample mixing. Second strand synthesis was performed using UT2-HA followed by purification. Fragments were then amplified using FAM-ST1,ROX-ST2, and UT2. Aliquots were collected, separated by CE, and analyzed to generate the electropherograms shown. C: Total rat brain RNA (1.75 �g) wasprocessed by STAR as described in B except that for PCR amplification NED-ST2 replaced ROX-ST2 (top panel) and VIC-ST2 replaced ROX-ST2 (bottom). D:Comparison of signal generated from 50 and 75 pg of input transcript. Two analytes were prepared in a background of 1.75 �g of total rat brain RNA. Analyte1 was spiked with 50 pg of VS31 and 50 pg of VS85. Analyte 2 was spiked with 50 pg of VS31 and 75 pg of VS85. Each analyte was reverse transcribed separatelyusing sample-specific sequence ST1dT14-VN or ST2dT14-VN generating cDNAs that were sequence tagged with either ST1 (analyte 1) or ST2 (analyte 2). ResultingcDNAs from both analytes were pooled and purified. Second strand was then synthesized using a forward sequence tag, UT2-HA. Again, excess primer wasremoved. Multiplex real-time PCR was performed using three primers, UT2, ROX-labeled ST1, and FAM-labeled ST2. Aliquots were taken from cycles 20 to 43and analyzed by CE.
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labeled ST1 and ROX-labeled ST2, and forward primerUT2.
As proof of principle, we analyzed the amplificationprofiles of two identical rat brain RNA samples that hadbeen spiked with VS31, an artificial transcript of definedsize. Analysis of electropherograms demonstrates that,as expected, a similar series of transcripts are amplifiedincluding the spiked transcript (Figure 5B; top, asterisk).Unexpectedly, we find that ROX labeling introduced a2-base shift in each of the analyzed fragments. To dem-onstrate that residual peaks were not due to nonspecificpriming of spiked VS31, the experiment was repeatedwithout spiking (Figure 5B, bottom). As shown, the VS31fragment is absent while background peaks amplifiedfrom the total RNA background are still present.
Further experiments explored the feasibility of usingother fluorophore combinations. Two identical samples oftotal RNA were subjected to STAR analysis as describedabove, except that for PCR amplification ROX-labeledST2 was replaced with NED-labeled ST2 (Figure 5C, top)or VIC-labeled ST2 (Figure 5C, bottom). We found thatboth fluorophores NED and VIC could be used in com-bination with FAM and, unlike ROX, do not produce a shiftin peak size. Using these labels, we observed completelyoverlapping peaks (Figure 5C).
To determine the differentiating ability of STAR usingsequence tags, experiments were performed to analyzevarying quantities of spiked artificial transcripts in thebackground of total rat brain RNA. Fifty pg of VS31 wasspiked into two rat RNA samples, whereas amount ofVS85 differed 1.5-fold in these samples. Analysis of theamplification plot (Figure 5D) demonstrated that, as ex-pected, equal amounts of target RNA were amplified withthe same kinetics. At the same time, a 1.5-fold differencein the level of RNA transcript could be easily detected byamplification kinetics or threshold cycle measurement.
Diagnostic Applications of STAR Technology
For diagnostic purposes, a one-step reaction is the pre-ferred method because it minimizes manipulations thatcan lead to errors or contamination of samples. We se-lected five primer pairs for SARS-CoV, each specific forone of the major genes.23,24 These primer pairs wereevaluated in a multiplex STAR assay using a plasmidlibrary spanning the SARS-CoV genome. Primers detect-ing the Rep1B and S genes that showed the best ampli-fication efficiencies were selected for a one-step multi-plex RT-PCR protocol. During this process we found thatreplacing Taq polymerase with Vent(Exo�) polymerasesignificantly improved the detectability of PCR products(data not shown). This may be due to better thermosta-bility of Vent polymerase and its lack of 3� exonucleaseactivity that may result in slow degradation of 5�-labeledprimers and PCR products.25 Because Vent polymerasehad not been previously used in one-step RT-PCR reac-tions, we optimized the assay conditions to be compati-ble with Vent.
Detection of SARS-CoV RNA Using STARTechnology
Using our optimized assay, both S and Rep1B genetargets were detected from 500,000 to 5 copies usingSARS-CoV RNA isolated from infected cultured Vero cells(data not shown). The assay was further validated usingmock clinical samples created by serial dilution of puri-fied SARS-CoV RNA into purified RNA samples obtainedfrom uninfected donors (Figure 6). The amount of SARS-CoV RNA spiked into normal human RNA isolated fromstool or sputum was in the range of 5.6 to 56,000 or 2 to200,000 copies per reaction, respectively. UnspikedRNAs served as negative controls. One-step multiplexRT-PCR was performed on each of the samples andanalyzed by the STAR protocol. Amplification curves andcycle threshold plots are shown for one of the multiplexedgenes, S, detected from stool and sputum RNA. Similarresults were obtained for Rep1B (data not shown). In stoolsamples, 5.6 copies were easily detected (Figure 6A)whereas 20 copies were detected from sputum (Figure6B). Although we did not detect SARS-CoV RNA at two
Figure 6. Detection of purified SARS-CoV RNA spiked into human biologicalsamples. Samples were 10-fold serially diluted from their original concentra-tion to 10 copies per reaction and amplified by one-step multiplex RT-PCRSTAR protocol for the S and Rep1B genes. Aliquots from cycles 20 to 43 wereanalyzed by CE to generate amplification curves. Data for the S gene areshown for stool-derived samples (A) [200,000 (filled squares), 20,000(open squares), 2000 (filled triangles), 200 (filled circles), and 20 (opencircles) copies] or sputum-derived samples (B) [56,000 (filled squares),5600 (open squares), 560 (filled circles), 56 (open circles), and 5.6(filled triangles) copies]. Insets: CT versus copy number graphs derivedfrom data in A and B.
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copies in sputum samples, rather than lack of sensitivity,this may reflect the statistical absence of viral RNA in thereaction. CT plots show good amplification efficiencies forthe S gene (100%) whether performed in a background ofhuman sputum or stool RNA (Figure 6, insets). Quantita-tive differences in CT values for sputum and stool reflectdifferences in RT efficiencies between RNA samples de-rived from different tissues.
SARS-CoV Detection from Blinded Clinical Samples
To further validate STAR, true clinical samples fromseveral tissue sources showing variable titers of SARSvirus were supplied as blinded samples and assessed forthe presence of SARS-CoV. Results of the STAR assaywere then compared with those obtained using theRoche LightCycler quantification kit. Both SARS-CoV tar-get sequences, S and Rep1B, were correctly detected infour tissue samples (intestine, lymph node, spleen, andthroat swab) obtained from an individual who had con-tracted SARS and subsequently died from the disease.Quantitative RT-PCR indicated that these RNA samplescontained 39,900 to 162 viral RNA copies/�l. Cyclethresholds calculated for both Rep1B and S showed goodreproducibility for each of the tissues (data not shown).Four blinded control samples were negative as assessedby STAR and the Roche LightCycler quantification kit.
Discussion
We have developed a novel PCR-based, highly accurate,and reliable method for multiplexed analysis of nucleicacids suitable for gene expression studies and clinicaldiagnostics. This method combines the quantitative ca-pacity of real-time PCR with the vast separating power ofCE allowing simultaneous measurement of dozens ofDNA or RNA targets in a single reaction. Integration ofPCR and CE for multiplexed assays was originally sug-gested as end-point PCR amplification followed by anal-ysis and quantification of the amplified targets by CE inwhich each target DNA was identified based on ampliconlength.26 This approach was successfully applied togene expression analysis22,27 and diagnostic applica-tions28–31 using end-point PCR detection. However, real-time PCR is preferred to endpoint PCR due to betterprecision and broader dynamic range of quantitativemeasurements.32 Li and colleagues33 furthered the tech-nology demonstrating that parallel single gene PCR re-actions terminated after successive cycles and analyzedby CE results in amplification curves for real-time analysisof a single target. STAR technology extends this conceptto enable real-time PCR algorithms for quantification ofmultiple targets within a single reaction. Rather than set-ting up separate PCR reactions for each aliquot, STARmonitors PCR amplification by withdrawing an aliquotfrom the same reaction tube after each successive cycle.This arrangement decreases the total volume required forPCR, avoids variability caused by nonuniform thermocy-cling across PCR plates, and permits simpler design of
an analytical instrument (for example by using directelectrokinetic transfer from PCR tube to the capillary).
A direct comparison of STAR to the two current stan-dards for gene expression analysis, TaqMan and SYBRshowed that STAR’s ability to quantify gene transcriptswas equal to these methods. We find that PCR efficien-cies observed for each of the methods are within therange of experimental error (Table 2) and are likely toreflect the different fluorescent labeling methods used.We have previously found that amplification efficienciescan be differentially affected by covalent modifications ofprimers (data not shown). TaqMan assays can also showvariable PCR efficiencies based on nucleotide composi-tion, hybridization kinetics, and probe positioning.34 Thisresult is in good agreement with other multiplex real-timePCR methods in which efficiency of multiplex PCR couldbe optimized to the level of single gene amplification.35
We also observed consistently lower threshold cycles forSTAR assays than for TaqMan and SYBR assays. Thisobservation can be partly explained by differences in thedetection methods used (laser-induced fluorescencereading using ABI 3100 Prism instrument, versus charge-coupled device camera readings on the Bio-Rad ICyclerreal-time PCR system). However, there is a fundamentaldifference in the sensitivity of STAR and other opticalreal-time PCR methods due to reaction mechanisms.STAR directly measures fluorescence of the PCR prod-uct, whereas TaqMan and SYBR assess a secondaryreaction that relies on an interaction between the PCRproduct and a signal-generating probe or dye, respec-tively. It is therefore not unexpected that methods basedon the detection of primary PCR products are more sen-sitive than methods based on secondary signal-generat-ing probe or dye intercalation.
STAR has several other advantages over current real-time PCR methods, especially for multiplex applications.First, STAR tolerates a much broader range of ampliconsize (50 to 1000 bases with current ABI CE systems) thancurrent real-time PCR methods (generally limited to 70 to200 bases) enabling efficient choice of primer pairs thatare compatible based on shared physical properties andcomposition. For targets that show high sequence vari-ability such as HIV-1,36 a broader scope of options forprimer design is available. Second, because detectiondoes not require probe hydrolysis, all restrictions relatedto the probe design (probe length and composition, dis-tance to PCR primers) do not influence primer selection.Third, STAR technology has the potential to analyze hun-dreds of targets per reaction due to discrimination ofDNA fragments both by size and fluorophore color. Cur-rent real-time PCR methods are limited to four targets perreaction. Finally, as specific PCR products are well sep-arated from potential nonspecific PCR products (for ex-ample, primer-dimers), primer design criteria for STARprotocols can be relaxed.
The potential of STAR for larger scale multiplexing isillustrated by real-time amplification of 12 genes in asingle RT-PCR reaction. We have also developed anin-house single nucleotide polymorphism assay thatmultiplexes 20 alleles within a single reaction (data notshown). To our knowledge, these are the first examples
452 Garcia et alJMD October 2005, Vol. 7, No. 4
of real-time PCR of more than 10 genes simultaneously.Importantly, development of this assay required noprimer optimization because the first set selected bycommonly used primer design software was success-fully implemented. Moreover, multiplexing with STAR isonly limited by the separation power of CE, which canseparate hundreds of DNA fragments differing by asingle nucleotide in size. However, expanding STARmultiplex assays to include dozens to hundreds oftargets using gene-specific primers can result in se-vere background due to primer interactions and non-specific amplification. The alternate priming strategiesoutlined that result in incorporation of sequence tagsdramatically simplifies the PCR reaction by reducingthe number of primers required, thereby decreasingbackground amplification.
Unlike current multiplexed RT-PCR methods, STAR canalso detect and quantify changes in gene expression pat-terns even if the identity of the gene is unknown. MultiplexedRT-PCR methods using TaqMan or Molecular Beacons re-quire a priori sequence knowledge of each target assayed.Using sequence tags and random hexamers as outlined inFigure 5, we have observed simultaneous real-time ampli-fication of 150 different DNA fragments using STAR fordirect transcriptional profiling (Figure 5, B and C) and havefound that the patterns obtained from total rat brain RNA arereproducible (data not shown). Zhong and Yeung37 havesimilarly found that tissue-specific patterns of cDNAs aregenerated when RNA is reverse transcribed using randomhexamers. We anticipate that a genetic fingerprint can bedefined for various tissues for each second strand primerused. Each fragment can be predicted from availablegenomic sequence data or identified by sequencing todefine the expressed transcriptome for each of the tissues.Because the real-time quantitative algorithm used in STARprovides better quantification of small differences as well asa much broader dynamic range allowing broadly ex-pressed transcripts to be analyzed, this database couldserve as a diagnostic tool because the presence/absenceand expression level of each fragment would be known.Novel amplicons may represent overexpressed genes,products of gene fusion, or trans-splicing, alternativelyspliced genes, or genes derived from pathogens. The useof multiple fluorophores also allows comparison of at leasttwo samples during transcriptional profiling suggesting thatSTAR would be comparable to microarrays with the addedadvantage of identifying new genetic transcripts.
Besides applications for gene expression analyses,multiplexed assays using STAR protocols offer consider-able advantages for molecular diagnostics. In addition tothe obvious consideration of cost, multiplexed assaysrequire less sample material to be withdrawn from pa-tients, and much simpler logistics regarding laboratorytests. As a first step in the development of multiplexeddiagnostic assays based on STAR technology we de-signed an assay for quantitative measurement of SARS-CoV. This assay was developed as a one-step RT-PCR todetect and quantify SARS-CoV RNA throughout a broadconcentration, ranging from several viral copies up toseveral hundred thousand viral copies per reaction. In ablinded study, our assay correctly identified both SARS-
CoV target sequences from tissue samples obtained froma SARS-infected individual with titers ranging from 39,900to 162 viral copies per reaction without showing anyfalse-positives. The STAR assay for SARS-CoV detectionused simultaneous amplification of several target se-quences from the SARS-CoV genome to confirm speci-ficity of the assay especially in the context of complexclinical samples. This assay serves as a prototype fordiagnostic tests in which a single clinical sample can beassessed for the presence of several pathogens. Alter-natively, targeting several regions of the pathogen ge-nome may be necessary for detection of rapidly mutatingorganisms because use of a single pair of specificprimers may lead to false-negatives due to mutation ofthat site. This could be particularly important in the casewhen mutations are associated with virulence or drugresistance.38
STAR technology could be used for many immediateapplications, most of which require multiplexing in therange of several dozen genes rather than hundreds.Such applications may include quantitative analysis ofgene sets comprising a molecular signature for a biolog-ical process, effects of drug treatment on gene expres-sion, and transcriptional signatures of disease conditions(identified in transcriptional profiling studies using DNAmicroarrays). An added benefit of the STAR approach isits scalability and flexibility regarding multigene assays.Any STAR assay can be easily updated by addition orsubtraction of particular primer pair(s) as long as there isno overlap in amplicon size. Finally, the STAR protocol isadaptable to high-throughput automation that in itssimplest form would include a dispensing thermocyclercoupled to CE.
These considerations will multiply for analyses re-quired for biodefense applications such as: medical sur-veillance; automated air, food, and water monitoring; andagricultural surveillance. All of these applications shouldhave the capacity to identify and to measure multiplepathogens in a single sample with high sensitivity in acost-effective manner. The potential of STAR could berealized through the direct integration of PCR with ultra-fast CE separation. As a first step in STAR automation, weassembled a breadboard of the dispensing thermocyclerto facilitate accurate sampling of PCR reaction. This sys-tem is based on the integration of a robotic fluid dis-penser and a remotely operated thermocycler that will bedescribed elsewhere. A specialized instrument that inte-grates PCR thermocycling, sample dispensing, and cap-illary-based separation is currently under development.Aside from instrument design, STAR development wouldalso require a comprehensive bioinformatics packagespanning multiplex primer design to management ofgene expression data generated by STAR. As a first step,we developed a prototype software analysis program thatfacilitates the conversion of electropherogram data intoreal-time amplification plots thereby reducing the timerequired to analyze STAR-generated data from days tominutes. The features and benefits described abovemake the STAR approach a natural candidate for devel-opment of a broad array of multiplex assays.
STAR Technology for Molecular Diagnostics 453JMD October 2005, Vol. 7, No. 4
Acknowledgments
We thank Drs. Randall Carpenter, Kazumi Shiosaki, LeonCooper, Mark Bear, Mel Epstein, and Stan Rose for sup-porting this project at Sention; Dr. Edward Yeung for helpfuldiscussions regarding capillary electrophoresis; Tim Clark,Marco Ocana, and Georgios Asteris for development ofsoftware tools for data analysis; Dr. Vivian Cheung for sta-tistical analysis of nucleotide sequences; and Lisa Ng andSe Thoe Su Yun for SARS-CoV sample preparation.
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