- Email: [email protected]
High throughput, nanoliter quantitative PCR
Drug Discovery Today: Technologies
Vol. 2, No. 3 2005
Editors-in-Chief Kelvin Lam – Pfizer, Inc., USA Henk Timmerman – Vrije Universiteit, The Netherlands DRUG DISCOVERY
TODAY
TECHNOLOGIES
Target identification
High throughput, nanoliter quantitative PCR Colin Brenan*, Tom Morrison BioTrove Inc., 12 Gill Street, Suite 4000, Woburn, MA 01810, USA
The recent completion of the human genome sequence has increased the need for high throughput quantitative transcription analysis. Quantitative PCR is an alternative to microarrays for accurate and precise expression analysis with single transcript copy sensitivity. A review of current research in miniaturized, high throughput qPCR suggests this technique will soon be a viable option to hybridization microarrays for largescale genetic analyses. Introduction The survival, growth and differentiation of a cell in normal and diseased states is reflected in altered patterns of gene expression and the ability to quantitate transcript levels of specific genes is central to research into gene function. The recent completion of the human genome sequence and the emergence of molecular medicine has increased the need for higher throughput techniques to quantitate levels of RNA across many hundreds of genes and thousands of samples. Faced with this challenge, oligonucleotide [1,2] and cDNA [3] microarrays have emerged as the leading quantitative tool for analyzing transcription levels in many thousands of genes in a sample simultaneously [4]. Despite this apparent success, it is well established that microarray data are fraught with errors from a variety of sources [5,6] with the greatest contribution from the platform itself [7]. The quantitative polymerase chain reaction (qPCR) is the standard by which the quality of microarray data is judged and validated [8]. PCR is a high-fidelity process for replicating a specific DNA sequence at levels down to a single molecule. *Corresponding author: C. Brenan ([email protected]) 1740-6749/$ ß 2005 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddtec.2005.08.017
Section Editors: Steve Gullans – RxGen, Inc., New Haven, CT, USA Robert Zivin – Johnson and Johnson, New Brunswick, NJ, USA This analytical versatility has made PCR an indispensable component of many bioanalytical methods and ubiquitous in modern biology. A solution-phase assay carried out in 96or 384-well microplates, scaling PCR to achieve higher throughputs with conventional technology is neither cost effective nor efficient. Consequently, it is therefore natural to consider if a larger number of PCR assays could be implemented simultaneously in smaller reaction volumes without compromising data quality. In other words, is it possible to combine the parallelism of a microarray with the quantification, sensitivity, dynamic range and specificity of qPCR to create a new high throughput platform for transcription analysis?
Quantitative PCR PCR is a temperature-modulated, enzymatic amplification for in vitro exponential replication of a nucleic acid sequence (target) defined by a pair of oligonucleotide sequences (primers) hybridized to their sequence complement [9]. Raising the solution temperature to 958C melts the double-stranded DNA (dsDNA) into two single strands, lowering the temperature to 50–658C hybridizes primers to the single strands and increasing the temperature to around 728C synthesizes complementary strands of template by action of the Taq polymerase. Repeating this temperature cycle C times leads to NC copies of the target region (amplicon) as: NC ¼ No ð1 þ EÞC
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where No is the starting copy number of target dsDNA and E is the amplification efficiency [10]. Inclusion of an intercalating fluorescent dye like SYBR GreenTM or an oligonucleotide probe with an attached quenched fluorescent dye, such as TaqmanTM [11] or molecular beacons [12], generates a fluorescent signal proportional to the increase in amplified product with the number of temperature cycles. Kinetic or realtime PCR quantifies the number of template DNA copies by calibration of the fluorescent amplification signal with copy number [13]. When the amplification signal reaches a level significantly above background, the fluorescence or cycle threshold (CT) is recorded and converted into template copy number based on a calibrated standard curve for that gene. RNA quantification requires reverse transcription of RNA into cDNA before application of the real-time PCR method.
qPCR miniaturization: challenges and benefits Miniaturization of PCR reaction volumes to less than a microliter lowers consumption of expensive reagents and decreases amplification times from the reduced thermal mass of the reaction volume. It confers flexibility in selection of a strategy to scale analytical throughput, either by a fast serial or parallel array processing approach. These attributes must be balanced against the requirement the quality of data from a low volume PCR system equal that from larger volume reactions, typically 5–10 mL, in a microplate. A crucial challenge in reaching this level of performance is the physical isolation of the reaction volumes to prevent evaporation and fluidic crosstalk between adjacent containers during thermal cycling and loading of sample and primers. Equally important are facile methods for liquid transfer of primer pairs, samples and PCR reagents between individual microcontainers and wells in a microplate without crosscontamination. Another factor impacting PCR assay quality in reduced volumes is the increased surface area-to-volume ratio. Surface interactions biasing PCR chemistry and kinetics can be mitigated by engineered coatings of the wetted surface for minimized reactivity or reformulation of the PCR by inclusion of compensating surface blocking agents. Paradoxically, qPCR often consumes the same amount of sample independent of reaction volume. This is based on the requirement to keep templates at a high enough starting copy number to avoid Poisson statistical noise (typically >300 starting copies) that could degrade assay dynamic range, precision and accuracy. Typically, SYBR Green qPCR requires a sample concentration of <2 ng/mL and for Taqman PCR, <50 ng/mL. There is an upper limit stemming from qPCR failure from too high a starting template concentration. Smaller volumes benefit from faster thermal cycling than larger volumes because the high surface area-to-volume ratio facilitates rapid heat transfer. Fabrication of microwell structures in high thermal conductivity, low specific heat materials like silicon enable shorter thermal cycle times than those 248
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in standard microplate thermoplastics having a low thermal conductivity and a high specific heat.
PCR arrays and microdevices Strategies for increasing PCR throughput and minimizing cost typically follow a twofold approach: decrease the reaction volume required for amplification and increase the number of reactions performed over a given time. Continuous-flow PCR devices utilize etched microchannels with fixed temperature zones to reduce reaction volumes to submicroliter levels and analyze PCR products by hybridization followed by electrochemical detection [14], fluorescence detection [15] or electrophoretic separation with fluorescent detection [16]. The LabChipTM 90 System from Caliper Life Sciences (http://www.caliperls.com/) is a commercial microfluidic version of [16] as an alternative to slab gel electrophoresis for automated quantitative DNA sizing and concentration. A common characteristic of these lab-on-achip devices is their high degree of functional integration that is accomplished by leveraging semiconductor microfabrication techniques to construct the microchannel network, miniature heaters, valves and sensors needed to execute the workflow of nucleic acid extraction, purification and amplification from a sample in an automated, miniaturized device. However, a key drawback in continuous-flow systems is the limited sample throughput and potential for crosscontamination that can result from processing and analyzing samples through a common microchannel. Techniques for decreasing PCR cycle time include the work by Huhmer et al. [17] who employed infrared radiation to achieve an order of magnitude faster heating and cooling rates than conventional thermal cycler blocks in a 160 nL PCR reaction contained in a glass microcapillary. Nagai et al. [18] pushed the limits further by amplifying a single 100 bp test fragment by rapid thermal cycling (168C/s) an array of 85 pL wells etched in a silicon plate, sealed with a glass slide and mineral oil and moved by hand between hot plates at different temperatures. A massively parallel strategy addresses both throughput and miniaturization simultaneously by creation of microplates with smaller volume wells in higher density arrays. One early attempt resulted in the first portable analytical qPCR instruments for viral detection [19,20] incorporating 50 mL chambers micromachined in silicon. Interestingly, significantly improved reaction fidelity was observed by lining the reaction chamber with polypropylene, the same material used in PCR microplates. This advance was eclipsed a few years later when Nagai et al. [21] reported amplifying a single 100 bp sequence in ten thousand 86 pL pyramidal chambers etched in a silicon wafer with a well density of 40 mm2. Hydrophilic coating of the wetted silicon with the addition of bovine serum albumin as a surface blocking agent greatly improved the PCR reaction. Although each reaction chamber contained the same ampli-
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fied target sequence after thermal cycling, concern over crosscontamination during amplicon recovery resulted in the unwieldy protocol of first evaporating the solvent through a selectively permeable membrane prior to resolubilizing the amplicon for recovery. In a subsequent report, Matsubara demonstrated detection of three genes with Taqman PCR in human genomic DNA using a lower density silicon array (1.2 mm2) with 40 nL wells and an improved nanoliter dispensing system to deposit the Taqman primer-probes into different wells. The number of template copies in each well was determined by measuring end point fluorescence and calculating copy number from a standard curve relating PCR end point fluorescence to the number of template copies in the well. In an ensuing article, Matusbara extended this approach to detect five gene targets in Escherichia coli genomic DNA by Taqman PCR in 40 nL reaction volumes with a detectable limit of 0.4 copies of DNA target per well [22,23]. Kalinina applied a similar scheme prior to Matsubara’s work to determine the number of template molecules in a diluted sample by counting the number of positive PCRs in a set of replicate reactions. This approach was confirmed in experiments involving single copy detection of human beta actin template with Taqman PCR in a 10 nL end-point reaction inside a glass microcapillary. Of interest was the potential for isolation of the targeted template by sample dilution enabling highly specific, single molecule detection in a complex mixture [24–26]. A picofluidic implementation of this technique was recently reported by Daridon [27] at the Fluidigm Corporation (www.fluidigm.com) where single copies of a target template were detected in a high background-to-target ratio of 30,000:1 by massively partitioning the sample into ten thousand 1 pL chambers by an array of miniature valves. This ability for parallel processing and analysis of a large number of single copy reactions is a potential enabler of digital PCR as originally described by Vogelstein et al. [28]. Reported by the Cytonix Corporation (www.cytonix.com) is a novel microfluidic device where the PCR takes place in an array of microdroplets formed between two plates, where at least one of the plates has a hydrophilic pattern that defines the droplet array. Introduction of an immiscible fluid between the plates controls the formation of isolated droplets in the wetted regions [29]. An impressive increase in PCR throughput and miniaturization was reported by Leamon et al. [30] at the 454 Life Sciences Inc. (www.454.com) with their high density PicoTiterPlateTM (480 mm2) in which they demonstrated three hundred thousand 39.5 pL simultaneous solid-phase DNA amplifications with asymmetric PCR reactions for clonal DNA amplification. Researchers at Norchip AS (http://www.norchip.com/) are pursuing an alternative to temperature-cycled PCR in a nanofluidic system for future applications in clinical diagnostics. Nucleic acid sequence-based amplification (NASBA) is an
Drug Discovery Today: Technologies | Target identification
isothermal, real-time technique specifically designed for amplification of RNA [31]. The Norchip group originally showed equivalence between a 50 nL and a 20 mL real-time NASBA reaction in the detection of a single-stranded DNA positive control for HPV 16 [32]. The group recently reported NASBA detection of target HPV and SiHa cell line sequences in a cyclic olefin copolymer microfluidic chip with ten 80 nL reaction chambers [33]. The primary advantage of isothermal RNA amplification is simplicity and significantly lower instrument cost but this could be offset by the potential complexity in assay design and lower sensitivity compared with temperature-cycled PCR. The technologies developed to-date for qPCR rely primarily on end-point PCR for quantitative analysis and have limited capability in handling liquids at volumes less than a microliter. The qPCR system developed by BioTrove Inc. (http:// www.biotrove.com/) addresses many of these problems and is based on the concept of the through-hole array [34–36]. Given the name OpenArrayTM, the device is a rectilinear array of three thousand, seventy-two 33 nL through-holes micromachined in a stainless steel platen the size of a standard microscope slide (25 mm 75 mm). Sixty-four throughholes are grouped in 48 subarrays spaced on a 4.5 mm pitch equal to the wells of a 384-well microplate (Fig. 1). The platen surface is chemically modified with a proprietary process to make the inside surface of each channel hydrophilic and the outside surface hydrophobic. The differential hydrophilic– hydrophobic coating facilitates precise loading (CV < 2%) and retention in isolation of fluid in every channel. The workflow for implementing qPCR in the OpenArray starts with transferring individual primer pairs stored in 384well microplates to individual through-holes with an array of 48 slotted pins manipulated by a four-axis robot in an environmentally controlled chamber. Once a platen is fully populated with primer pairs, the solvent is evaporated in a controlled manner leaving the primers immobilized in a matrix on the inside surface of each through-hole. The arrays are stored in this state, ready for sample addition. Next, 48 previously prepared cDNA samples (32 ng/mL) are mixed with off-the-shelf qPCR reagents for SYBR Green PCR (Roche LightCycler Mastermix) and loaded into each sub-array (one sample per sub-array) with a 48 pipette tip dispensing device called the Array-in-ArrayTM. A plug of UV curable epoxy seals the array with an immiscible fluid to prevent evaporation during thermal cycling. Three encased arrays are placed on the flat block of an imaging thermal cycler (NT Cycler) programmed to implement a real-time PCR protocol. An assay-centric software environment enables acquisition and processing of fluorescence amplification and melt curves, including CT calculation and copy number estimate, from 9216 real-time PCR analyses in less than 4 h. The accuracy of qPCR in the OpenArray is determined by measuring the CT as a function of template starting copy www.drugdiscoverytoday.com
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Figure 1. Anatomy of the OpenArrayTM. A stainless steel platen the size of a standard microscope slide with 3072 micromachined through-holes of 33 nL each. There are 48 groups of 64 through-holes each on a 4.5 mm pitch. Proprietary chemical modifications make the inside surface of each through-hole hydrophilic and the outside surface hydrophobic.
Figure 2. (a) Fluorescence amplification curves from seven 10-fold amplicon dilutions decreasing amplicon number from 107 to 10 copies per throughhole of Cyc A amplicon in a 33 nL PCR reaction volume. Note the delayed, non-specific amplification after cycle number 35 for the no template control through-holes. (b) PCR efficiency and precision for the Cyc A primer set. (c) Fluorescent image of an OpenArray with 3072 different primer pairs showing PCR amplification at cycle 27.
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Figure 3. (a) Interarray correlation of CT for 3072 RT-PCR reactions in the OpenArray with human liver cDNA. (b) Intermicroplate correlation of CT with 10 mL RT-PCR in a 384-well microplate with 384 primer pair subset from the OpenArray and human liver cDNA.
number over seven log dilutions (107 to 10 starting copies per through-hole, 16 replicates per dilution) and a no template control (Fig. 2a). As expected, the amplification curves shift as a function of starting copies and when the CT of these curves are plotted against the number of starting copies (Fig. 2b), seven logs of dynamic range was observed with a PCR efficiency, estimated by the slope of this line, of nearly 100% and a 0.45 CT precision at 1000 copies. Figure 2c is a SYBR Green fluorescence image of an OpenArray loaded with human liver cDNA (Stratagene) equivalent to 82 pg of mRNA per through-hole after 27 cycles with a different BioTrove designed and validated primer pair in each through-hole. Different levels of fluorescence from each through-hole correspond to different transcript levels in the original sample. CT correlation in OpenArray replicates (Fig. 3a) shows the data is reproducible and compares favorably with the CTs calculated from 10 ml qPCR microplate replicates performed with 384 primer pairs selected from a set of 3072 primer pairs previously run in the OpenArray (Fig. 3b).
Conclusions Recent completion of the human genome sequence and emergence of molecular medicine has stimulated interest in finding ways to improve the cost effectiveness of qPCR
at higher analytical throughputs. Clearly, PCR miniaturization is one attractive strategy because it allows a larger number of analyses to be performed serially or in parallel faster and at lower cost because of reduced reagent consumption and decreased amplification times. One research direction has been towards lab-on-a-chip microdevices fully integrating the necessary steps for implementing a PCR-based assay. A high level of workflow integration in a miniature, automated device enables stand-alone applications in environmental sensing and diagnostics but the serial nature of sample processing in these devices greatly limits their throughput relative to the demands of functional genomics research. An array of qPCR reactions is an attractive alternative because it combines the parallelism of microarrays with the quantification, sensitivity, dynamic range and specificity of qPCR. Although research has shown the feasibility of qPCR arrays, practical solutions to a number of basic problems have not been found. These include (i) robust liquid handling for simple interfacing with microplates, (ii) ways for keeping the reaction volumes in isolation during thermal cycling to prevent assay degradation from crosstalk, (iii) configuring the reaction environment to make the assay performance independent of reaction volume and (iv) increasing the sample www.drugdiscoverytoday.com
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Table 1. Comparative assessment of the principal commercially-available technologies for high throughput, quantitative transcription analysis Microarrays
qPCR
qPCR
Name of specific type of technology
DNA microarray
Low density arrays (96- & 384-well microplates)
High density arrays (OpenArrayTM)
Names of specific technologies with associated companies and company websites
GeneChipTM Array Affymetrix Inc. www.affymetrix.com Human Genome CHG44a & b Agilent Inc. www.agilent.com
7300, 7500, 7900 Real-time PCR Systems Applied Biosystems Inc. www.appliedbiosystems.com LightCycler 2.0 & 480 Roche Applied Science www.roche-applied-science.com
OpenArray BioTrove Inc. www.biotrove.com
Pros
Large number of targets per samples Low cost per data point
High precision and accuracy Single molecule sensitivity and large dynamic range Low sample consumption
High precision and accuracy Single molecule sensitivity and large dynamic range Short sample preparation and turnaround time High number of targets per sample High sample throughput Low cost per data point
Short sample preparation and turnaround time
High sample consumption Long sample preparation and turnaround time Low sample throughput
Cons
References
Weis et al. [7] Larkin et al. [8]
High cost per data point Low number of targets per samples Low sample throughput Bustin, 2002
concentration to maintain PCR sensitivity and dynamic range with decreasing reaction volume. Of the technologies reviewed, only the OpenArray technology provides solutions for most, if not all, of these problems. A comparative assessment of DNA microarray, qPCR in microplates with qPCR in the OpenArray (Table 1) shows the OpenArray compares favorably with microarrays in throughput and cost and to qPCR in a microplate in sensitivity and sample throughput. Clearly systems like the OpenArray will meet the requirements of researchers interested in increasing the number of sample analyses without compromising data quality. Although high density qPCR arrays might one day displace microarrays in transcription analysis, a more probable scenario is that microarrays will be used for discovery applications where large numbers of genes will be analyzed in a few samples and systems like the OpenArray will be the platform for hypothesis-driven research where the transcription of a small to moderate number of selected genes will be quantified across a large number of samples. What does the future hold? One could imagine systems like the OpenArray eventually displacing microplates for most routine biochemical assays, thereby achieving the same level of ubiquity as microplates. A higher level of workflow integration in either serial, lab-on-a-chip formats or parallel, array formats is to be anticipated, particularly within the context of miniaturized instrumentation for portable, stand-alone biosensing. An intriguing possibility is to combine in one platform the capability for simultaneous protein and nucleic acid mea252
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Moderate sample consumption
Brenan et al. [36]
surement from a sample or an array of samples, resulting in a concordant data set spanning the transcription of the genetic code into protein information. This could be an attractive system for system biology studies looking to understand
Links Hong, J.W. and Quake, S.R. (2003) Integrated nanoliter systems. Nat. Biotechnol. 21, 1179–1183: http://www.nature.com/nbt/journal/v21/ n10/full/nbt871.html PCR efficiency and quantitation of gene expression: http://www.gene-quantification.de/efficiency.html Articles of quantitative PCR analysis methods and techniques: http://depts.washington.edu/genomelb/ Quantitative%20PCR%20analysis%20methods.html
Related articles Verpoote, E. et al. (2003) Microfluidics meets MEMS. Proc. IEEE, 91, 930–953 Kricka, L. et al. (2003) Microchip PCR. Anal. Bioanal. Chem. 377, 820– 825 Weigl, R. et al. (2003) Lab-on-a-chip for drug development. Adv. Drug Deliv. Rev. 55, 349–377 Bustin, S.A. (2002) Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J. Mol. Endocrinol. 29, 23–29 de Mello, A.J. (2001) DNA amplification: does ‘‘small’’ really mean ‘‘efficient’’? Lab Chip 1, 24N–29N
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Outstanding issues Limiting development of high throughput, low volume qPCR technology:
Simple, robust liquid handling interface with microplates. Reaction volume isolation during thermal cycling and liquid handling. Assay miniaturization without compromising data quality. Higher sample concentrations.
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large-scale functional relationships between DNA, RNA and protein.
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References 1 Pease, A.C. et al. (1994) Light generated oligonuleotide arrays for rapid DNA sequence analysis. Proc. Natl. Acad. Sci. U S A 11, 5022–5026 2 Lipshutz, R.J. et al. (1995) Using oligonucleotide probe arrays to access genetic diversity. Biotechniques 19, 442–447 3 Schena, M. et al. (1995) Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 5235, 368–369 4 Gershon, D. (2002) Micorarray technology: an array of opportunities. Nature 416, 885–891 5 Murphy, D. (2002) Gene expression studies using microarrays: principles, problems and prospects. Adv. Physiol. Educ. 26, 256–270 6 Petersen, D. et al. (2005) Three microarray platforms: an analysis of their concordance in profiling gene expression. BMC Genomics 6, 63 7 Weis, B.K. et al. (2005) Standardizing global gene expression analysis between laboratories and across platforms. Nat. Methods 5, 351–356 8 Larkin, J.E. et al. (2005) Independence and reproducibility across microarray platforms. Nat. Methods 5, 337–343 9 Mullis, K. et al. (1986) Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harbor Symp. Quant. Biol. 51 (Pt 1), 263–273 10 Rutledge, R.G. et al. (2003) Mathematics of quantitative kinetic PCR and the application of standard curves. Nucleic Acids Res. 31, 93 11 Holland, P.M. et al. (1991) Detection of specific polymerase chain reaction product by utilizing the 50 ! 30 exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. U S A 88, 7276–7280 12 Fang, X. et al. (2000) Molecular beacons: novel fluorescent probes. Anal. Chem. 23, 747A–753A 13 Higuchi, R. et al. (1993) Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology 9, 1026–1030 14 Liu, R.H. et al. (2004) Self-contained, fully integrated biochip for sample preparation, polymerase chain reaction amplification, and DNA microarray detection. Anal. Chem. 76, 1824–1831 15 Lee, D-S. et al. (2004) Bulk-micromachined submicroliter-volume PCR chip with very rapid thermal response and low power consumption. Lab Chip 4, 401–407
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31 32 33 34 35 36
Lagally, E.T. et al. (2001) Single-molecule DNA amplification and analysis in an integrated microfluidic device. Anal. Chem. 3, 565–570 Huhmer, A.F.R. et al. (2000) Noncontact infrared-mediated thermocycling for effective polymerase chain reaction amplification of DNA in nanoliter volumes. Anal. Chem. 72, 5507–5512 Nagai, H. et al. (2001) High-throughput PCR in silicon based microchamber array. Biosens. Bioelectron. 16, 1015–1019 Northrup, M.A. et al. (1998) A miniature analytical instrument for nucleic acids based on micromachined silicon reaction chambers. Anal. Chem. 70, 918–922 Ibrahim, M.S. et al. (1998) Real-time microchip PCR for detecting singlebase differences in viral and human DNA. Anal. Chem. 70, 2013–2017 Nagai, H. et al. (2001) Development of a microchamber array for picoliter PCR. Anal. Chem. 73, 1043–1047 Matsubara, Y. et al. (2004) On-chip nanoliter-volume multiplex TaqMan polymerase chain reaction from a single copy based on counting fluorescence released from microchambers. Anal. Chem. 21, 6434–6439 Matsubara, Y. et al. (2005) Microchamber array based DNA quantification and specific sequence detection from a single copy via PCR in nanoliter volumes. Biosens. Bioelectron. 20, 1482–1490 Kalinina, O. et al. (1997) Nanoliter scale PCR with TaqMan detection. Nucleic Acids Res. 10, 1999–2004 Brown, J.F. et al. (2000) Method of sampling, amplifying and quantifying segment of nucleic acid, polymerase chain reaction assembly having nanoliter-sized sample chambers, and method of filling assembly. USP 6,143,496 Brown, J.F. et al. (2002) Method of sampling, amplifying and quantifying segment of nucleic acid, polymerase chain reaction assembly having nanoliter-sized sample chambers, and method of filling assembly. USP 6,391,559 B1 Daridon, A. (2005) Picofluidic digital PCR. Quantitative PCR. La Jolla, CA, USA Vogelstein, B. et al. (1999) Digital PCR. Proc. Natl. Acad. Sci. U S A 16, 9236– 9241 Silver, J. et al. (1999) Controlled formation of low-volume liquid pillars between plates with a lattice of wetting patches by use of a second immiscible fluid. J. Colloid Interface Sci. 219, 81–89 Leamon, J. et al. (2003) A massively parallel PicoTiterPlateTM based platform for discrete picoliter-scale polymerase chain reactions. Electrophoresis 24, 3769–3777 Compton, J. (1991) Nucleic acid sequence-based amplification. Nature 350, 91–92 Gulliksen, A. et al. (2004) Real-time nucleic acid sequence-based amplification in nanoliter volumes. Anal. Chem. 76, 9–14 Gulliksen, A. et al. (2005) Parallel nanoliter detection of cancer markers using polymer microchips. Lab Chip 4, 416–420 Brenan, C.J.H. et al. (2000) BioMEMS applied to the development of cellbased bioassay systems. SPIE Proc. 3912, 76–87 Kanigan, T.K. et al. (2000) Living chips for drug discovery. SPIE Proc. 3926, 172–180 Brenan, C.J.H. et al. (2002) A massively parallel microfluidics platform for storage and ultra high throughput screening. SPIE Proc. 4626, 560–569
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Vol. 2, No. 3 2005
Editors-in-Chief Kelvin Lam – Pfizer, Inc., USA Henk Timmerman – Vrije Universiteit, The Netherlands DRUG DISCOVERY
TODAY
TECHNOLOGIES
Target identification
High throughput, nanoliter quantitative PCR Colin Brenan*, Tom Morrison BioTrove Inc., 12 Gill Street, Suite 4000, Woburn, MA 01810, USA
The recent completion of the human genome sequence has increased the need for high throughput quantitative transcription analysis. Quantitative PCR is an alternative to microarrays for accurate and precise expression analysis with single transcript copy sensitivity. A review of current research in miniaturized, high throughput qPCR suggests this technique will soon be a viable option to hybridization microarrays for largescale genetic analyses. Introduction The survival, growth and differentiation of a cell in normal and diseased states is reflected in altered patterns of gene expression and the ability to quantitate transcript levels of specific genes is central to research into gene function. The recent completion of the human genome sequence and the emergence of molecular medicine has increased the need for higher throughput techniques to quantitate levels of RNA across many hundreds of genes and thousands of samples. Faced with this challenge, oligonucleotide [1,2] and cDNA [3] microarrays have emerged as the leading quantitative tool for analyzing transcription levels in many thousands of genes in a sample simultaneously [4]. Despite this apparent success, it is well established that microarray data are fraught with errors from a variety of sources [5,6] with the greatest contribution from the platform itself [7]. The quantitative polymerase chain reaction (qPCR) is the standard by which the quality of microarray data is judged and validated [8]. PCR is a high-fidelity process for replicating a specific DNA sequence at levels down to a single molecule. *Corresponding author: C. Brenan ([email protected]) 1740-6749/$ ß 2005 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddtec.2005.08.017
Section Editors: Steve Gullans – RxGen, Inc., New Haven, CT, USA Robert Zivin – Johnson and Johnson, New Brunswick, NJ, USA This analytical versatility has made PCR an indispensable component of many bioanalytical methods and ubiquitous in modern biology. A solution-phase assay carried out in 96or 384-well microplates, scaling PCR to achieve higher throughputs with conventional technology is neither cost effective nor efficient. Consequently, it is therefore natural to consider if a larger number of PCR assays could be implemented simultaneously in smaller reaction volumes without compromising data quality. In other words, is it possible to combine the parallelism of a microarray with the quantification, sensitivity, dynamic range and specificity of qPCR to create a new high throughput platform for transcription analysis?
Quantitative PCR PCR is a temperature-modulated, enzymatic amplification for in vitro exponential replication of a nucleic acid sequence (target) defined by a pair of oligonucleotide sequences (primers) hybridized to their sequence complement [9]. Raising the solution temperature to 958C melts the double-stranded DNA (dsDNA) into two single strands, lowering the temperature to 50–658C hybridizes primers to the single strands and increasing the temperature to around 728C synthesizes complementary strands of template by action of the Taq polymerase. Repeating this temperature cycle C times leads to NC copies of the target region (amplicon) as: NC ¼ No ð1 þ EÞC
[1] www.drugdiscoverytoday.com
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where No is the starting copy number of target dsDNA and E is the amplification efficiency [10]. Inclusion of an intercalating fluorescent dye like SYBR GreenTM or an oligonucleotide probe with an attached quenched fluorescent dye, such as TaqmanTM [11] or molecular beacons [12], generates a fluorescent signal proportional to the increase in amplified product with the number of temperature cycles. Kinetic or realtime PCR quantifies the number of template DNA copies by calibration of the fluorescent amplification signal with copy number [13]. When the amplification signal reaches a level significantly above background, the fluorescence or cycle threshold (CT) is recorded and converted into template copy number based on a calibrated standard curve for that gene. RNA quantification requires reverse transcription of RNA into cDNA before application of the real-time PCR method.
qPCR miniaturization: challenges and benefits Miniaturization of PCR reaction volumes to less than a microliter lowers consumption of expensive reagents and decreases amplification times from the reduced thermal mass of the reaction volume. It confers flexibility in selection of a strategy to scale analytical throughput, either by a fast serial or parallel array processing approach. These attributes must be balanced against the requirement the quality of data from a low volume PCR system equal that from larger volume reactions, typically 5–10 mL, in a microplate. A crucial challenge in reaching this level of performance is the physical isolation of the reaction volumes to prevent evaporation and fluidic crosstalk between adjacent containers during thermal cycling and loading of sample and primers. Equally important are facile methods for liquid transfer of primer pairs, samples and PCR reagents between individual microcontainers and wells in a microplate without crosscontamination. Another factor impacting PCR assay quality in reduced volumes is the increased surface area-to-volume ratio. Surface interactions biasing PCR chemistry and kinetics can be mitigated by engineered coatings of the wetted surface for minimized reactivity or reformulation of the PCR by inclusion of compensating surface blocking agents. Paradoxically, qPCR often consumes the same amount of sample independent of reaction volume. This is based on the requirement to keep templates at a high enough starting copy number to avoid Poisson statistical noise (typically >300 starting copies) that could degrade assay dynamic range, precision and accuracy. Typically, SYBR Green qPCR requires a sample concentration of <2 ng/mL and for Taqman PCR, <50 ng/mL. There is an upper limit stemming from qPCR failure from too high a starting template concentration. Smaller volumes benefit from faster thermal cycling than larger volumes because the high surface area-to-volume ratio facilitates rapid heat transfer. Fabrication of microwell structures in high thermal conductivity, low specific heat materials like silicon enable shorter thermal cycle times than those 248
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in standard microplate thermoplastics having a low thermal conductivity and a high specific heat.
PCR arrays and microdevices Strategies for increasing PCR throughput and minimizing cost typically follow a twofold approach: decrease the reaction volume required for amplification and increase the number of reactions performed over a given time. Continuous-flow PCR devices utilize etched microchannels with fixed temperature zones to reduce reaction volumes to submicroliter levels and analyze PCR products by hybridization followed by electrochemical detection [14], fluorescence detection [15] or electrophoretic separation with fluorescent detection [16]. The LabChipTM 90 System from Caliper Life Sciences (http://www.caliperls.com/) is a commercial microfluidic version of [16] as an alternative to slab gel electrophoresis for automated quantitative DNA sizing and concentration. A common characteristic of these lab-on-achip devices is their high degree of functional integration that is accomplished by leveraging semiconductor microfabrication techniques to construct the microchannel network, miniature heaters, valves and sensors needed to execute the workflow of nucleic acid extraction, purification and amplification from a sample in an automated, miniaturized device. However, a key drawback in continuous-flow systems is the limited sample throughput and potential for crosscontamination that can result from processing and analyzing samples through a common microchannel. Techniques for decreasing PCR cycle time include the work by Huhmer et al. [17] who employed infrared radiation to achieve an order of magnitude faster heating and cooling rates than conventional thermal cycler blocks in a 160 nL PCR reaction contained in a glass microcapillary. Nagai et al. [18] pushed the limits further by amplifying a single 100 bp test fragment by rapid thermal cycling (168C/s) an array of 85 pL wells etched in a silicon plate, sealed with a glass slide and mineral oil and moved by hand between hot plates at different temperatures. A massively parallel strategy addresses both throughput and miniaturization simultaneously by creation of microplates with smaller volume wells in higher density arrays. One early attempt resulted in the first portable analytical qPCR instruments for viral detection [19,20] incorporating 50 mL chambers micromachined in silicon. Interestingly, significantly improved reaction fidelity was observed by lining the reaction chamber with polypropylene, the same material used in PCR microplates. This advance was eclipsed a few years later when Nagai et al. [21] reported amplifying a single 100 bp sequence in ten thousand 86 pL pyramidal chambers etched in a silicon wafer with a well density of 40 mm2. Hydrophilic coating of the wetted silicon with the addition of bovine serum albumin as a surface blocking agent greatly improved the PCR reaction. Although each reaction chamber contained the same ampli-
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fied target sequence after thermal cycling, concern over crosscontamination during amplicon recovery resulted in the unwieldy protocol of first evaporating the solvent through a selectively permeable membrane prior to resolubilizing the amplicon for recovery. In a subsequent report, Matsubara demonstrated detection of three genes with Taqman PCR in human genomic DNA using a lower density silicon array (1.2 mm2) with 40 nL wells and an improved nanoliter dispensing system to deposit the Taqman primer-probes into different wells. The number of template copies in each well was determined by measuring end point fluorescence and calculating copy number from a standard curve relating PCR end point fluorescence to the number of template copies in the well. In an ensuing article, Matusbara extended this approach to detect five gene targets in Escherichia coli genomic DNA by Taqman PCR in 40 nL reaction volumes with a detectable limit of 0.4 copies of DNA target per well [22,23]. Kalinina applied a similar scheme prior to Matsubara’s work to determine the number of template molecules in a diluted sample by counting the number of positive PCRs in a set of replicate reactions. This approach was confirmed in experiments involving single copy detection of human beta actin template with Taqman PCR in a 10 nL end-point reaction inside a glass microcapillary. Of interest was the potential for isolation of the targeted template by sample dilution enabling highly specific, single molecule detection in a complex mixture [24–26]. A picofluidic implementation of this technique was recently reported by Daridon [27] at the Fluidigm Corporation (www.fluidigm.com) where single copies of a target template were detected in a high background-to-target ratio of 30,000:1 by massively partitioning the sample into ten thousand 1 pL chambers by an array of miniature valves. This ability for parallel processing and analysis of a large number of single copy reactions is a potential enabler of digital PCR as originally described by Vogelstein et al. [28]. Reported by the Cytonix Corporation (www.cytonix.com) is a novel microfluidic device where the PCR takes place in an array of microdroplets formed between two plates, where at least one of the plates has a hydrophilic pattern that defines the droplet array. Introduction of an immiscible fluid between the plates controls the formation of isolated droplets in the wetted regions [29]. An impressive increase in PCR throughput and miniaturization was reported by Leamon et al. [30] at the 454 Life Sciences Inc. (www.454.com) with their high density PicoTiterPlateTM (480 mm2) in which they demonstrated three hundred thousand 39.5 pL simultaneous solid-phase DNA amplifications with asymmetric PCR reactions for clonal DNA amplification. Researchers at Norchip AS (http://www.norchip.com/) are pursuing an alternative to temperature-cycled PCR in a nanofluidic system for future applications in clinical diagnostics. Nucleic acid sequence-based amplification (NASBA) is an
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isothermal, real-time technique specifically designed for amplification of RNA [31]. The Norchip group originally showed equivalence between a 50 nL and a 20 mL real-time NASBA reaction in the detection of a single-stranded DNA positive control for HPV 16 [32]. The group recently reported NASBA detection of target HPV and SiHa cell line sequences in a cyclic olefin copolymer microfluidic chip with ten 80 nL reaction chambers [33]. The primary advantage of isothermal RNA amplification is simplicity and significantly lower instrument cost but this could be offset by the potential complexity in assay design and lower sensitivity compared with temperature-cycled PCR. The technologies developed to-date for qPCR rely primarily on end-point PCR for quantitative analysis and have limited capability in handling liquids at volumes less than a microliter. The qPCR system developed by BioTrove Inc. (http:// www.biotrove.com/) addresses many of these problems and is based on the concept of the through-hole array [34–36]. Given the name OpenArrayTM, the device is a rectilinear array of three thousand, seventy-two 33 nL through-holes micromachined in a stainless steel platen the size of a standard microscope slide (25 mm 75 mm). Sixty-four throughholes are grouped in 48 subarrays spaced on a 4.5 mm pitch equal to the wells of a 384-well microplate (Fig. 1). The platen surface is chemically modified with a proprietary process to make the inside surface of each channel hydrophilic and the outside surface hydrophobic. The differential hydrophilic– hydrophobic coating facilitates precise loading (CV < 2%) and retention in isolation of fluid in every channel. The workflow for implementing qPCR in the OpenArray starts with transferring individual primer pairs stored in 384well microplates to individual through-holes with an array of 48 slotted pins manipulated by a four-axis robot in an environmentally controlled chamber. Once a platen is fully populated with primer pairs, the solvent is evaporated in a controlled manner leaving the primers immobilized in a matrix on the inside surface of each through-hole. The arrays are stored in this state, ready for sample addition. Next, 48 previously prepared cDNA samples (32 ng/mL) are mixed with off-the-shelf qPCR reagents for SYBR Green PCR (Roche LightCycler Mastermix) and loaded into each sub-array (one sample per sub-array) with a 48 pipette tip dispensing device called the Array-in-ArrayTM. A plug of UV curable epoxy seals the array with an immiscible fluid to prevent evaporation during thermal cycling. Three encased arrays are placed on the flat block of an imaging thermal cycler (NT Cycler) programmed to implement a real-time PCR protocol. An assay-centric software environment enables acquisition and processing of fluorescence amplification and melt curves, including CT calculation and copy number estimate, from 9216 real-time PCR analyses in less than 4 h. The accuracy of qPCR in the OpenArray is determined by measuring the CT as a function of template starting copy www.drugdiscoverytoday.com
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Figure 1. Anatomy of the OpenArrayTM. A stainless steel platen the size of a standard microscope slide with 3072 micromachined through-holes of 33 nL each. There are 48 groups of 64 through-holes each on a 4.5 mm pitch. Proprietary chemical modifications make the inside surface of each through-hole hydrophilic and the outside surface hydrophobic.
Figure 2. (a) Fluorescence amplification curves from seven 10-fold amplicon dilutions decreasing amplicon number from 107 to 10 copies per throughhole of Cyc A amplicon in a 33 nL PCR reaction volume. Note the delayed, non-specific amplification after cycle number 35 for the no template control through-holes. (b) PCR efficiency and precision for the Cyc A primer set. (c) Fluorescent image of an OpenArray with 3072 different primer pairs showing PCR amplification at cycle 27.
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Figure 3. (a) Interarray correlation of CT for 3072 RT-PCR reactions in the OpenArray with human liver cDNA. (b) Intermicroplate correlation of CT with 10 mL RT-PCR in a 384-well microplate with 384 primer pair subset from the OpenArray and human liver cDNA.
number over seven log dilutions (107 to 10 starting copies per through-hole, 16 replicates per dilution) and a no template control (Fig. 2a). As expected, the amplification curves shift as a function of starting copies and when the CT of these curves are plotted against the number of starting copies (Fig. 2b), seven logs of dynamic range was observed with a PCR efficiency, estimated by the slope of this line, of nearly 100% and a 0.45 CT precision at 1000 copies. Figure 2c is a SYBR Green fluorescence image of an OpenArray loaded with human liver cDNA (Stratagene) equivalent to 82 pg of mRNA per through-hole after 27 cycles with a different BioTrove designed and validated primer pair in each through-hole. Different levels of fluorescence from each through-hole correspond to different transcript levels in the original sample. CT correlation in OpenArray replicates (Fig. 3a) shows the data is reproducible and compares favorably with the CTs calculated from 10 ml qPCR microplate replicates performed with 384 primer pairs selected from a set of 3072 primer pairs previously run in the OpenArray (Fig. 3b).
Conclusions Recent completion of the human genome sequence and emergence of molecular medicine has stimulated interest in finding ways to improve the cost effectiveness of qPCR
at higher analytical throughputs. Clearly, PCR miniaturization is one attractive strategy because it allows a larger number of analyses to be performed serially or in parallel faster and at lower cost because of reduced reagent consumption and decreased amplification times. One research direction has been towards lab-on-a-chip microdevices fully integrating the necessary steps for implementing a PCR-based assay. A high level of workflow integration in a miniature, automated device enables stand-alone applications in environmental sensing and diagnostics but the serial nature of sample processing in these devices greatly limits their throughput relative to the demands of functional genomics research. An array of qPCR reactions is an attractive alternative because it combines the parallelism of microarrays with the quantification, sensitivity, dynamic range and specificity of qPCR. Although research has shown the feasibility of qPCR arrays, practical solutions to a number of basic problems have not been found. These include (i) robust liquid handling for simple interfacing with microplates, (ii) ways for keeping the reaction volumes in isolation during thermal cycling to prevent assay degradation from crosstalk, (iii) configuring the reaction environment to make the assay performance independent of reaction volume and (iv) increasing the sample www.drugdiscoverytoday.com
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Table 1. Comparative assessment of the principal commercially-available technologies for high throughput, quantitative transcription analysis Microarrays
qPCR
qPCR
Name of specific type of technology
DNA microarray
Low density arrays (96- & 384-well microplates)
High density arrays (OpenArrayTM)
Names of specific technologies with associated companies and company websites
GeneChipTM Array Affymetrix Inc. www.affymetrix.com Human Genome CHG44a & b Agilent Inc. www.agilent.com
7300, 7500, 7900 Real-time PCR Systems Applied Biosystems Inc. www.appliedbiosystems.com LightCycler 2.0 & 480 Roche Applied Science www.roche-applied-science.com
OpenArray BioTrove Inc. www.biotrove.com
Pros
Large number of targets per samples Low cost per data point
High precision and accuracy Single molecule sensitivity and large dynamic range Low sample consumption
High precision and accuracy Single molecule sensitivity and large dynamic range Short sample preparation and turnaround time High number of targets per sample High sample throughput Low cost per data point
Short sample preparation and turnaround time
High sample consumption Long sample preparation and turnaround time Low sample throughput
Cons
References
Weis et al. [7] Larkin et al. [8]
High cost per data point Low number of targets per samples Low sample throughput Bustin, 2002
concentration to maintain PCR sensitivity and dynamic range with decreasing reaction volume. Of the technologies reviewed, only the OpenArray technology provides solutions for most, if not all, of these problems. A comparative assessment of DNA microarray, qPCR in microplates with qPCR in the OpenArray (Table 1) shows the OpenArray compares favorably with microarrays in throughput and cost and to qPCR in a microplate in sensitivity and sample throughput. Clearly systems like the OpenArray will meet the requirements of researchers interested in increasing the number of sample analyses without compromising data quality. Although high density qPCR arrays might one day displace microarrays in transcription analysis, a more probable scenario is that microarrays will be used for discovery applications where large numbers of genes will be analyzed in a few samples and systems like the OpenArray will be the platform for hypothesis-driven research where the transcription of a small to moderate number of selected genes will be quantified across a large number of samples. What does the future hold? One could imagine systems like the OpenArray eventually displacing microplates for most routine biochemical assays, thereby achieving the same level of ubiquity as microplates. A higher level of workflow integration in either serial, lab-on-a-chip formats or parallel, array formats is to be anticipated, particularly within the context of miniaturized instrumentation for portable, stand-alone biosensing. An intriguing possibility is to combine in one platform the capability for simultaneous protein and nucleic acid mea252
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Moderate sample consumption
Brenan et al. [36]
surement from a sample or an array of samples, resulting in a concordant data set spanning the transcription of the genetic code into protein information. This could be an attractive system for system biology studies looking to understand
Links Hong, J.W. and Quake, S.R. (2003) Integrated nanoliter systems. Nat. Biotechnol. 21, 1179–1183: http://www.nature.com/nbt/journal/v21/ n10/full/nbt871.html PCR efficiency and quantitation of gene expression: http://www.gene-quantification.de/efficiency.html Articles of quantitative PCR analysis methods and techniques: http://depts.washington.edu/genomelb/ Quantitative%20PCR%20analysis%20methods.html
Related articles Verpoote, E. et al. (2003) Microfluidics meets MEMS. Proc. IEEE, 91, 930–953 Kricka, L. et al. (2003) Microchip PCR. Anal. Bioanal. Chem. 377, 820– 825 Weigl, R. et al. (2003) Lab-on-a-chip for drug development. Adv. Drug Deliv. Rev. 55, 349–377 Bustin, S.A. (2002) Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J. Mol. Endocrinol. 29, 23–29 de Mello, A.J. (2001) DNA amplification: does ‘‘small’’ really mean ‘‘efficient’’? Lab Chip 1, 24N–29N
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Outstanding issues Limiting development of high throughput, low volume qPCR technology:
Simple, robust liquid handling interface with microplates. Reaction volume isolation during thermal cycling and liquid handling. Assay miniaturization without compromising data quality. Higher sample concentrations.
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large-scale functional relationships between DNA, RNA and protein.
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