Gains in Sensitivity with a Device that Mixes Microarray Hybridization

Anal. Chem. 2002, 74, 6413-6417

Gains in Sensitivity with a Device that Mixes Microarray Hybridization Solution in a 25-µm-Thick Chamber Nils B. Adey,*,†,‡ Ming Lei,†,‡ Mike T. Howard,†,‡ John D. Jensen,†,‡ Debbie A. Mayo,†,‡ Darin L. Butel,†,‡ Steve C. Coffin,†,‡ Tom C. Moyer,† Devan E. Slade,†,‡ Mark K. Spute,†,‡ Angela M. Hancock,§ George T. Eisenhoffer,§ Brian K. Dalley,§ and Michael R. McNeely†,‡

BioMicro Systems, Inc., 1290 W, 2320 S, Suite D, Salt Lake City, Utah, 84119, and Huntsman Cancer Institute, Salt Lake City, Utah 84112

A microarray hybridization system that allows mixing in volumes comparable to those used by glass coverslips is presented. This system is composed of a disposable flexible lid that binds to 1 in. × 3 in. glass slides via an adhesive gasket, forming a uniform 25-µm-thick hybridization chamber. This chamber rests on a base unit for temperature control. The lid contains two air-driven bladders that continuously mix the hybridization fluid. Mixing enhances sensitivity from a typical microarray experiment 2-3-fold. Mixing is particularly effective at high spotted probe and low labeled target concentrations and overcoming local target depletion that occurs when homologous probes are spotted in close proximity. Mixing appears to be compatible with most hybridization conditions; however, mix versus no-mix control experiments should be performed. Also covered are a number of microfluidic issues related to manufacturing, filling, mixing, and packaging. DNA microarray analysis is the latest in a line of hybridizationbased techniques that query a nucleic acid sample.1 The microarray approach enables massive throughput and, therefore, has become a dominant technology with applications in a variety of fields such as gene expression profiling, DNA sequencing, and medical diagnostics. DNA microarrays are small “chips”, such as microscope slides, that contain thousands of discrete spots attached to their surface.2-5 Each spot is a unique DNA fragment or “probe” of known sequence. These microarrays are incubated * Corresponding author: (e-mail) [email protected]. † BioMicro Systems, Inc. ‡ Current addresses: (N.B.A.) Wasatch Bioconsulting, 2218 Emerson Ave., Salt Lake City, UT 84108. (M.L.) 454 Corp., Branford, CT 06450. (M.T.H.) Department of Human Genetics, Salt Lake City, UT 84112. (J.D.J.) Anteon Corp., Clearfield, UT 84015. (D.A.M.) Echelon Biosciences, Inc., Salt Lake City, UT 84108. (D.L.B.) Lumenis Corp., Salt Lake City, UT 84115. (S.C.C.) Embedded Systems Engineering, Layton, UT 84040. (D.E.S.) LT Communications, Salt Lake City, UT 84116. § Huntsman Cancer Institute. (1) Southern, E.; Mir, K.; Shchepinov, M. Nat. Genet. (Suppl.) 1999, 21, 5-9. (2) Brown, P. O.; Botstein, D. Nat. Genet. (Suppl.) 1999, 21, 33-37. (3) Pease, A. C.; Solas, D.; Sullivan, E. J.; Cronin, M. T.; Holmes, C. P.; Fodor, S. P. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 5022-5026. (4) Schena, M.; Shalon, D.; Davis, R. W.; Brown, P. O. Science 1995, 270, 467-470. (5) Shalon, D.; Smith, S. J.; Brown, P. O. Genome Res. 1996, 6, 639-645. 10.1021/ac026082m CCC: $22.00 Published on Web 11/15/2002

© 2002 American Chemical Society

with solutions containing unknown nucleic acid “targets” derived from a variety of sources such as a cellular extract or an environmental sample. Target nucleic acids that are complementary to probe nucleic acids hybridize and remain bound through subsequent washing steps. Typically, targets are covalently modified with fluorescent dyes prior to the hybridization step so microarray spots containing bound targets fluoresce when excited with a slide scanning device. The intensity of the fluorescence is an indication of the quantity of complementary target nucleic acid in the starting sample. A tremendous amount of effort has been expended to optimize microarray technology. Despite this, the most commonly used hybridization chamber is simply a glass coverslip or a second slide in what is known as a sandwich hybridization. The entire chamber is sealed in a humid container to reduce evaporation. These manual methods are proven and have favorable hybridization kinetics due to a low hybridization chamber volume and thus high target concentration.6 However, these methods have a number of inherent shortcomings. A major shortcoming is the lack of hybridization solution mixing.7-9 Without mixing, the number of target molecules available for hybridization is limited by diffusion. For a typical DNA molecule, this distance has been determined to be less than 1 mm,10 suggesting that, for any given spot on a 18 mm × 54 mm array, less than 0.3% of the available target has an opportunity for hybridization. If a target is in low abundance, it may become depleted near the complementary probe spot. Mixing should minimize localized target depletion, resulting in improved detection of low-abundance gene transcripts, a major obstacle when DNA microarrays are used.11,12 Other shortcomings (6) Chan, V.; Graves, D. J.; McKenzie, S. E. Biophys. J. 1995, 69, 2243-2255. (7) Freeman, W. M.; Robertson, D. J.; Vrana, K. E. Biotechniques 2000, 29, 1042-1055. (8) Steel, A.; Torres, M.; Hartwell, J.; Yu, Y.; Ting, N.; Hoke, G.; Yang, H. Microarray Biochip Technology; Eaton Publishing: Natick, MA, 2000; Chapter 5. (9) Watson, A.; Mazumder, A.; Stewart, M.; Balasubramanian, S. Curr. Opin. Biotechnol. 1998, 9, 609-614. (10) Worley, J.; Bechtol, K.; Penn, S.; Roach, D.; Hanzel, D.; Trounstine, M.; Barker, D. Microarray Biochip Technology; Eaton Publishing: Natick, MA, 2000; Chapter 4. (11) Duggan, D. J.; Bittner, M.; Chen, Y.; Meltzer, P.; Trent, J. M. Nat. Genet. (Suppl.) 1999, 21, 10-14. (12) Gerhold, D.; Rushmore, T.; Caskey, C. T. Trends Biochem. Sci. 1999, 24, 168-173.

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with manual methods include warped coverslips that lead to uneven volumes of hybridization solution above various spots and wicking or evaporation near the edges that lead to fluid loss and nonuniform hybridization solution concentrations. A number of commercial microarray hybridization systems exist that provide hybridization solution mixing. However, the hybridization chamber volumes on these systems are typically 3-fold or greater than in manual methods and therefore provide less favorable hybridization kinetics. Here, we describe the development of the microarray user interface (MAUI) hybridization system, a system that has the ability to continuously mix volumes similar to those used in manual methods. The MAUI system consists of a flexible plastic “lid” that binds to a microarray slide via an adhesive gasket and a controlled temperature base unit. This system was used to show hybridization solution mixing can deliver 2-3-fold gains in sensitivity (signal versus noise) in a typical cDNA microarray experiment. EXPERIMENTAL SECTION Bacteriophage λ PCR Amplicons. Standard PCR conditions were used to amplify the bacteriophage λ genome (GenBank accession number ××00906). For Figure 2A, identical probes and targets were amplified from the following: (A) 30537-30836; (B) 7131-7630; (C) 4021-4260; (D) 3721-3899; (E) (probe only) 5461-5820. For Figure 2B, 16 probes and 16 targets were amplified from the following: (pair 1) 5461-7353, 6853-7353; (pair 2) 28916-30611, 30211-30611; (pair 3) 2581-4097, 37214097; (pair 4) 21481-23078, 22683-23078; (pair 5) 27160-27725, 27541-28080; (pair 6) 40058-40686, 40501-41100; (pair 7) 31141-32157, 31971-32931; (pair 8) 12099-13141, 12957-14015. Target was labeled by including 0.1 mM Cy3 or Cy5 dCTP (Amersham Pharmacia) in the PCR reaction and reducing unlabeled dCTP concentration to 0.025 mM. PCR products were purified using the Concert Rapid PCR system (Life Technologies Catalog No. 11458-023), visualized by gel electrophoresis, and quantitated with either PicoGreen (Molecular Probes Catalog No. P-11495) or directly for Cy3 and Cy5. Microarray Hybridization Protocol Using λ PCR Products. Purified PCR products (25 ng/µL in 50% DMSO) were spotted using an Amersham BioSciences GEN III spotter (0.8 nL/spot) on glass microscope slides (Fisher Catalog No. 12-544-1) treated with (3-aminopropyl)methyldiethoxysilane (Sigma Catalog No. 09309). Using a modification of Hegde et al.,13 spotted microarray slides were incubated for 2 min in 95 °C water, dried with compressed nitrogen, incubated 2 h in 42 °C 5× SSC, 0.1% SDS, and 1% BSA, dipped 5× in water and once in 2-propanol, and again dried with compressed nitrogen. Labeled target and Cot-1 DNA (Life Technologies Catalog No. 15279; final hybridization concentration, 0.13 µg/µL) was denatured for 2 min at 95°C in 5× SSC) and then added to SSCD hybridization solution (5× SSC, 0.1% SDS, 5× Denhardt’s, 50% formamide). This solution was either loaded into a MAUI device as described below or placed on a slide (32 µL), covered with a Grace BioLabs 22 × 60 mm HybriSlip (Catalog No. HS60), sealed in a humidified Corning hybridization chamber, and incubated overnight in a 42 °C oven. The next day, the MAUI slides were disassembled in the A/D-jig that was (13) Hegde, P.; Qi, R.; Abernathy, K.; Gay, C.; Dharap, S.; Gaspard, R,; EarleHughes, J.; Snesrud, E.; Lee, N.; Quackenbush, J. Biotechniques 2000, 29, 548-562.

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submerged in 42 °C wash buffer 1. Next, all slides were agitated for 10 min in 42 °C wash buffer 1 (1× SSC, 0.2% SDS), 10 min in room temperature wash buffer 2 (0.1× SSC, 0.2% SDS), and two additional 4-min washes in room-temperature wash buffer 3 (0.1× SSC) and then dried with compressed nitrogen. The processed slides were scanned and quantitated using an Amersham Biosciences GEN III scanner and ArrayVision software (Imaging Research Inc.), the background was corrected, and the data were analyzed using Microsoft Excel. Spots produced by the first pin lie under one mixing bladder (the GEN III spotter cannot control array position) and, while not obviously harmed, were excluded from the analysis. Microarray Hybridization Protocol for Human Cell Line RNA. A 6912 PCR product probe array (Sequence Verified Human cDNA clone set from InVitrogen) was duplicated on the left and right halves of each slide. Total RNA from RKO and HCT116 cells was isolated using Trizol reagent according to the manufacturer’s instructions (InVitrogen, Catalog No. 15596-026) and amplified using the RiboAmp kit (Arcturus Engineering, Catalog No. KIT0201). Amplified target RNA (aRNA) was labeled by either Cy3 (RKO) or Cy5 (HCT116) modified dCTP (Amersham BioSciences) by reverse transcription (Superscript II, InVitrogen). Labeled products were pooled, divided into 2-µg aliquots, dried in a SpeedVac, resuspended in water, diluted to 36 (sandwich hybridizations) or 60 µL (MAUI hybridizations; only 35-40 µL entered the MAUI lid because a syringe was used) in 1× final SSCD hybridization buffer (described above), denatured at 95 °C for 2 min, loaded, and then incubated overnight at 42 °C. Following hybridization, MAUI (see above) and sandwich hybridizations were disassembled in 42 °C wash buffer 1, incubated 10 min in 42 °C wash buffer 1, twice for 10 min in 42 °C wash buffer 2, and three times for 1 min in room temperature wash buffer 3. Slides were briefly rinsed in 0.02× SSC and then dried with compressed air. Scanning and data analysis was performed as described above. Filling and Sealing of the MAUI Lid. Hybridization solution was injected using the positive displacement Eppendorf Combitip syringe (Brinkmann, Catalog No. 022-26-595-4) controlled by the electronic Repeater Pro (Brinkmann, Catalog No. 022-46-117-6). Filling was facilitated by prewarming the hybridization solution to 42 °C to reduce viscosity and by using low molecular weight polymers such as the carrier DNA. Forty-five microliters of hybridization solution was slowly aspirated (to avoid air bubbles) into the Combitip, the tip inserted into the fill port with sufficient perpendicular downward force to ensure a tight seal but not to deform the tip or lid, and the solution injected into the lid. The outsides of both fill and vent ports were dried with paper towels and then sealed with adhesive disks. Adhesive nipples were attached over the air ports, air hoses applied that connect the nipples with the base unit, the air pump turned on, the cover closed to prevent target photobleaching, and the hybridization allowed to proceed overnight. A detailed protocol is available (www.biomicro.com). RESULTS AND DISCUSSION The MAUI microarray hybridization system was developed to mix hybridization solution volumes similar to those employed in manual methods. The hybridization chamber is created by attaching a disposable lid to a 1 in. × 3 in. glass microarray slide via a 25-µm-thick adhesive gasket that eliminates the need for a

Figure 1. (A) Drawing of the assembled MAUI lid on a microarray slide. The hybridization chamber accommodates up to a 20.5 mm × 55.0 mm microarray. (B) The MAUI lid is a four-layer laminate composed of (1) an adhesive gasket layer made from laminating adhesive that seals the lid to the slide and defines the hybridization chamber dimensions, (2) a diaphragm layer made from thin plastic sheet that forms the inner surface of the hybridization chamber, and (3) a bladder layer made from laminating adhesive that defines the diaphragm dimensions and bonds the diaphragm layer to the (4) main support layer, made from thicker plastic sheet which is the outer surface of the lid.

clamp. The chamber covers a rectangular array up to 20.5 mm × 55 mm with a 35-40-µL layer of fluid (Figure 1A). Proper alignment of the lid with the slide is achieved using an Assembly Disassembly jig (A/D-jig); a firm seal is achieved by braying (rubbing) the top of the lid. Once attached, the slide and lid are placed in slots in the base unit. The base unit is essentially a large aluminum block that rests in a standard hot block heater. The lid is a four-layer adhesive laminate made from stock materials, which simplifies uniform manufacturing (Figure 1B). The upper three layers create two mixing bladders that are powered by a pneumatic system also located in the base unit. The pneumatic system pressurizes one air bladder while evacuating the other, creating a bulk movement of hybridization fluid across the microarray. After a set pause time, the pneumatic system is reversed, which drives the hybridization fluid in the opposite direction. This constitutes a basic mixing cycle. The pneumatic system is composed of an air compressor (a vacuum source is created by the compressor intake) connected to two three-way solenoid valves. All three are independently operated by an embedded microcontroller and an EEPROM, which are time-based and programmed via PC-based serial port communications software. This system can accommodate complex mixing schemes. Additional details will be available when patent applications are published (ep.espacenet.com). Hybridization solution is injected into the chamber through a small cylindrical port in the top of the lid using an electronic positive displacement pipetting device. This device reduces noninjected hybridization solution to less than 10 µL and rarely traps air bubbles in the chamber because solution is pushed in more rapidly than capillary action can pull it. Outgassing of the plastic lid can also create air bubbles; this problem is avoided by

vacuum packing the lid for at least 4 days prior to use in a humidified container to prevent damage to the adhesive. The thickness uniformity of the fluid layer was assessed by adding Bromophenol Blue to the hybridization solution. Both the MAUI lid and sandwich hybridizations create very uniform fluid layers, in contrast to thin glass or plastic coverslips, which are often warped (not shown). Nonuniform fluid layers can result in nonuniform hybridization signals. Clear hybridization solution was subsequently injected to create a dye/no-dye boundary in the middle of the lid. When the pneumatic system was activated, the boundary was observed to move ∼10 mm with every mix cycle. While microarray signals are enhanced using a mixing cycle frequency as low as once every 3 h, the greatest signal enhancements were obtained using frequencies of less than 1 min (data not shown). Mixing cycles shorter than 5 s are inefficient because the viscous hybridization solution does not have adequate time to respond. Using 5-s mixing cycles, the dye/no-dye lid described above became homogeneous in ∼1 h (data not shown). This result was somewhat unexpected because the fluid movement within the chamber was assumed to be laminar and therefore the fluid should remain heterogeneous. Our observations suggest that homogeneity is achieved due to the flexible nature of the lid. When a mixing bladder is inflated, the viscous fluid forced from beneath the bladder causes the portion of the lid adjacent to the bladder to bulge and thus most of the fluid appears to travel down the center of the slide. However, when the bladder is evacuated, the fluid pulled under the bladder causes the lid to collapse and thus most of the fluid appears to travel along the edges of the slide. This creates what appears to be four circular currents, one in each quadrant of the lid, that may enhance mixing (not shown). The lid is removed from the slide by utilizing the A/D-jig. To prevent drying of the slide, which can create high background, the jig is immersed in wash solution during removal of the lid. If the lid, slide, and wash solution have equilibrated to 42 °C, the adhesive gasket will remain bound to the lid because at this temperature the adhesive has a higher affinity for the diaphragm material than for the glass slide. It is possible to recover used hybridization solution prior to lid disassembly. This is done by injecting additional hybridization solution (without nucleic acids or other molecules above 10 kDa) into the fill port forcing the used hybridization solution out the vent port. This solution can be collected from the top of the lid, reconcentrated in a microfiltration column, and reused. Microarray experiments were performed to determine whether hybridization solution mixing improves results. The first series of experiments utilized PCR to produce specific quantifiable products for both the immobilized probes and the labeled targets. Five nonhomologous probes, all spotted at high concentration, were hybridized with four complementary targets, each at a different concentration. Compared to nonmixed MAUI and coverslip hybridization chambers, mixed hybridization solution produced up to a 3-fold gain in sensitivity (Figure 2A). At the lowest target concentration, only the mixed MAUI hybridization chamber produced a reliable signal, suggesting that mixing might allow the detection of otherwise undetectable gene expression. At higher target concentrations, little to no significant sensitivity gain is realized. This is likely because local target concentrations are approaching available probe concentrations that would minimize Analytical Chemistry, Vol. 74, No. 24, December 15, 2002

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Table 1. Sensitivity Gains from Hybridization Solution Mixing expt

density of identical spots

concn probe (amol/spot)

mix/no mixb

1 1 2 2

high (8×)a low (1×) high (8×) low (2×)

2-20 2-20 100 100

2.5 ( 0.5 1.6 ( 0.2 7.9 ( 1.2 3.3 ( 1.4

a Number of identical spots within 1 mm. b Average of identical probe signals (background corrected) from mixed slides divided by not-mixed slides. Uncertainty is one standard deviation of 2-8 slides, each containing 3-13 different probes.

Figure 2. Microarray hybridization experiments using bacteriophage λ PCR products showing that hybridization solution mixing significantly improves sensitivity from low-abundance labeled targets. (A) The following quantity of labeled targets were used (fmol/slide): A, 22; B, 1.1; C, 0.10; D, 0.0094. Every microarray spot contained ∼0.1 fmol (100 amol) probe. Error bar represents one standard deviation of 22 identical microarray spots that were arranged as adjacent pairs, each pair being well separated from the other 10 pairs. (B) The legend indicates the mixing conditions and volume of hybridization solution used. One microliter contains ∼0.8 fmol of each of eight pairs of targets. One member of each pair is labeled with Cy3, the other with Cy5 (see Experimental Section). The fractional values plotted on the Y axis are calculated as follows: the Cy3 or Cy5 signal derived from given labeled target at the indicated conditions was divided by the maximum Cy3 or Cy5 signal for that particular labeled target at all experimental conditions tested (median signals from eight identical and well-separated spots were used in these calculations). Each point shown in the graph is the median fractional value from the set of eight pairs of targets; the error bars represent one standard deviation. The error bars were generally symmetric about each point, and thus for clarity, only the upper or lower is shown. One microliter of labeled target corresponds to 2.5 amol within 1 mm of a given spot, the estimated distance a typical target molecule diffuses during an overnight incubation. Less than 2.5 amol/µL is probably available for hybridization due to competition from the soluble complementary PCR product strand.

local target depletion. To investigate how the spotted probe concentration affects mixing, a second experiment was performed that used 16 different PCR amplicons for both the labeled targets and the spotted probes. The results (Figure 2B) show that when a target is limiting (32 amol/spot), mixing can enhance microarray 6416 Analytical Chemistry, Vol. 74, No. 24, December 15, 2002

Figure 3. Microarray hybridization experiments using human cDNAs also showing that mixing increases sensitivity. The X axis represents 6912 spot positions that were sorted based on the median Cy3 signal from all six microarrays. Because of high slide-to-slide signal variability at any given spot, the plotted Cy3 intensity is a median of a 100-spot window. This window was then moved by one spot and the process repeated to create a total of 6912 plotted intensity values for each microarray. Analysis of Cy5 signals produced similar results (data not shown). The relatively weak signal of the nonmixed MAUI versus the sandwich hybridizations could be a result of target dilution; a less efficient loading system was used (see Experimental Section).

signals, but when probe is limiting (2 amol/spot), mixing has no significant benefit, presumably because local target depletion does not occur. This experiment underscores the importance of spotting sufficient probe. In the course of these experiments, we observed that mixing provides greater sensitivity gains if identical spots are clustered (Table 1). This was not due to an improvement in absolute signal but to relief of clustering-induced signal suppression when hybridization solution is not mixed; presumably, identical spot clustering enhances local target depletion. It can be difficult to ensure that homologous probes are well separated on DNA microarrays, especially large probe sets that lack complete sequence information. Hybridization solution mixing can minimize artifacts caused by unintentional clustering of homologous probes. The second series of microarray experiments utilized human cell line cDNA as the labeled target and 6912 different commercially available cloned cDNAs as the probe sets to test a typical microarray expression system. The results (Figure 3) were generated by sorting all 6912 spot positions based on the median Cy3 signal from six different microarrays: two sandwich hybridizations and four MAUI hybridizations, two with and two without mixing. While all six microarrays have similar background levels, the microarrays that were subject to hybridization solution mixing

show rapid signal gains at a lower spot position and clearly produce stronger signals above spot position 4500. The relative gain in signal intensity of the mixed versus the not-mixed MAUI chambers is similar to the data in Figure 2A, suggesting mixing is operating in the same manner. Additional experiments demonstrated that two-day hybridizations in the presence of mixing produced even greater gains in sensitivity (data not shown). A third series of microarray experiments investigated whether certain experimental conditions are incompatible with hybridization solution mixing. The first experiment utilized 18 PCR labeled targets and 52 PCR product probes that ranged from 65 to 100% sequence identity, 135 bp to nearly 2000 bp in length and 75 bp to over 1000 bp of complementary overlap. While more than 40fold differences in signal were observed between different probes and the same target, these differences were the same in mixed and unmixed slides, which suggests that mixing does not produce a sequence-specific differential effect. This slide set was also used to examine slide coating and hybridization buffer constituents. No chamber leakage or mixing-specific effects were observed using five different types of commercially available slides or those described in the Experimental Section, demonstrating that, in general, slide coatings are not an issue. However, some brands of commercial hybridization buffers occasionally reduced signal intensity from a subset of probes when subject to mixing. The magnitude of this effect was variable and may have correlated with methods of use. The SSCD hybridization solution used in these studies consistently produced enhanced signals when subject to mixing. However, all hybridization conditions should be validated using mix versus no-mix experiments. This report covers the development and testing of the MAUI microarray hybridization system. The adhesive laminate design of the hybridization chamber is simple yet very effective. The chamber requires less than half the volume (per surface area) of other microarray hybridization systems with mixing potential, allowing for improved sensitivity. The chamber lid can be manufactured from stock materials, which benefits thickness

uniformity. The air bladder system is energy efficient compared with vibration-induced mixing, which minimizes temperature considerations, and once sealed, does not introduce bubbles, which can be a problem with fluid pumping systems. The bladder dimensions can be readily modified giving additional control over mixing rates. The adhesive gasket provides positive sealing, resulting in minimal fluid loss from overnight incubations at elevated temperatures. In addition, the developmental process identified an effective method to fill a low-profile, high-surface area chamber, described how a flexible chamber can enhance mixing, and showed that vacuum packing will prevent plastic outgassing and subsequent bubble formation. This system was used to investigate the benefits of microarray hybridization solution mixing. If sufficient probe is spotted, mixing enhances sensitivity 2-3-fold in a typical microarray experiment and to a greater extent if homologous probes are clustered in a small area of the slide. The data suggest that improved sensitivity results because mixing relieves local target depletion that may occur in static hybridization solution conditions. The MAUI hybridization system is simple, inexpensive, and easy to maintain making it ideal for smaller laboratories with irregular use cycles. ACKNOWLEDGMENT The authors thank Dale Emery and Robert Raleigh for developmental assistance and assembly of the instrument and lid; Dr. Eleanor Goodall and Cory Heidelberger for intellectual property work; Dr. William Pagels, Scott Romney, and Katie McDaniel for business and market development; Shelly Kaufman and Tiffani Farnell for administrative work; and Dr. Ann Greig for manuscript review. Evaluations with the cDNA array were performed at the Huntsman Cancer Institute and was supported by NCI Grant P30 CA42014-15. Received for review August 27, 2002. Accepted October 17, 2002. AC026082M

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