Expression profiling by oligonucleotide microarrays spotted on coated polymer slides

Journal of Biotechnology 116 (2005) 125–134

Expression profiling by oligonucleotide microarrays spotted on coated polymer slides Peter S. Nielsen∗ , Helle Ohlsson, Carsten Alsbo, Morten S. Andersen, Sakari Kauppinen Department of Functional Genomics, Exiqon, Bygstubben 9, DK-2950 Vedbaek, Denmark Received 9 July 2004; received in revised form 24 September 2004; accepted 11 October 2004

Abstract We have developed a ready-to-spot polymer microarray slide, which is coated with a uniform layer of reactive electrophilic groups using anthraquinone-mediated photo-coupling chemistry. The slide coating reduces the hydrophobicity of the native polymer significantly, thereby enabling robust and efficient one-step coupling of spotted 5 amino-linked oligonucleotides onto the polymer slide. The utility of the coated polymer slide in gene expression profiling was assessed by fabrication of spotted oligonucleotide microarrays using a collection of 5 amino-linked 70-mer oligonucleotide probes representing 96 yeast genes from Operon. Two-colour hybridizations with labelled cDNA target pools derived from standard grown and heat-shocked wild type yeast cells could reproducibly measure heat shock induced expression of seven different heat shock protein (HSP) genes. Moreover, the observed fold changes were comparable to those reported previously using spotted cDNA arrays and high-density 25-mer oligonucleotide arrays from Affymetrix. The low hybridization signals obtained from the
SSA4 mutant cDNA target, together with the high signal detected in two-colour hybridizations with heat-shocked wild type yeast relative to the
SSA4 mutant strain implies that unspecific binding of cDNA target to the SSA4-specific 70-mer oligonucleotide probes is negligible. Combined, our results indicate that the coated polymer microarray slide represents a robust and cost-effective array platform for pre-spotted oligonucleotide arrays. © 2004 Elsevier B.V. All rights reserved. Keywords: Anthraquinone; Photocoupling; Polymer slide; Microarray; Expression profiling

1. Introduction

∗ Corresponding author. Tel.: +45 45 65 04 41; fax: +45 45 66 18 88. E-mail address: [email protected] (P.S. Nielsen).

0168-1656/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2004.10.009

The power of DNA microarrays as experimental tools relies on the precise positioning of DNA probes on a solid support at high density and specific hybridization of the arrayed probes to their labelled comple-

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mentary targets. This allows massive parallel analyses of gene expression profiles, gene mutations and single nucleotide polymorphisms on a genomic scale (Fodor et al., 1991; Lockhart et al., 1996; Schena et al., 1996). Thus, DNA microarrays have become increasingly useful for addressing basic biological processes in a global fashion, as well as for disease diagnostics, toxicology, drug screening and development. Expression profiling by microarrays is typically carried out using either high-density oligonucleotide arrays fabricated in situ by photolithography (Fodor et al., 1991; Lipshutz et al., 1999) or spotted microarrays composed of oligonucleotides or PCR-amplified cDNAs printed onto glass slides (Schena et al., 1995). The utility of pre-spotted DNA oligonucleotide microarrays in gene expression profiling has been evaluated in several comprehensive studies (Dai et al., 2002; Kane et al., 2000; Ramakrishnan et al., 2002; Relogio et al., 2002; Wang et al., 2003). Compared to PCR products amplified from cDNA libraries, gene-specific oligonucleotides of 30–70 nt in length can be designed with optimised uniform hybridization properties and minimized crosshybridization using pertinent software. Besides controlled specificity, oligonucleotide microarrays enable detection of alternative mRNA splice isoforms and discrimination between highly homologous transcripts. Furthermore, the use of oligonucleotides provides a more cost-effective microarray platform avoid from clone tracking, PCR amplification and sequence verification associated with the fabrication of cDNA arrays. In a recent study, the feasibility of unmodified 70-mer oligonucleotides in expression profiling was assessed alongside with the corresponding PCR products printed onto the same glass slides (Wang et al., 2003). The results using both synthetic spike RNAs and complex cellular RNA target pools provided substantial evidence for comparable performance of the oligonucleotides and cDNAs for most of the genes studied (Wang et al., 2003). High quality spots having a uniform spot size, shape, and morphology, are crucial for successful DNA microarray analyses. The spot quality is influenced by multiple factors, including DNA concentration, the spotting buffer composition, and the relative humidity in the spotting facility. Currently glass is the preferred support for microarray fabrication due to its favourable optical characteristics, resistance to high temperature and low cost (Zammatteo et al., 2000). An inherent

property of glass slides however is their hydrophilic surface, resulting in a small contact angle for a DNAcontaining water droplet deposited onto the surface. This, in turn, may result in a relative large spot size and irregularly formed droplets. By comparison, a polymer support provides a more hydrophobic surface and consequently a high contact angle resulting in smaller droplet size. However, when printing with contact spotters, a highly hydrophobic surface would, most likely, present problems in array fabrication. Furthermore, deposited water droplets with a high contact angle have a small contact area between the surface and the liquid, and thus can easily be displaced. We have developed a ready-to-spot, injectionmoulded polymer microarray slide, which is coated with a uniform layer of reactive electrophilic groups using anthraquinone-mediated photo-coupling chemistry (Koch et al., 2000). The slide coating reduces the hydrophobicity of the native polymer significantly and enables robust and efficient one-step coupling of spotted 5 amino-linked PCR amplicons and oligonucleotides onto the polymer slide. In the present study, we show that the coated polymer slide is well suited for the fabrication of DNA oligonucleotide microarrays. In addition, we describe its utility in expression profiling by assessing the heat shock response in the yeast Saccharomyces cerevisiae using oligonucleotide microarrays composed of the Yeast Genome Sample Oligo Set from Operon printed on the polymer slide. We have chosen the yeast heat-shock response as a model system since it allowed comparison of our expression profiling results with data obtained from two yeast studies based on the use of either cDNA microarrays (Gasch et al., 2000) or Affymetrix GeneChips (Causton et al., 2001) in genome-wide expression analysis.

2. Materials and methods 2.1. Yeast cultures S. cerevisiae wild type (BY4741, MATa; his3
1; leu2
0; met15
0; and ura3
0) and the ∆SSA4 (MATa; his3
1; leu2
0; met15
0; ura3
0; and YER103w::kanMX4) mutant strain (EUROSCARF) were grown in YPD medium at 30 ◦ C until the A600 density of the cultures reached 0.8. Half of the cultures

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were collected by centrifugation and resuspended in 1 vol. of 40 ◦ C preheated YPD. Incubation was continued for an additional 30 min at 30 ◦ C or 40 ◦ C for the standard and heat-shocked cultures, respectively. Cells were harvested by centrifugation and stored at −80 ◦ C. 2.2. PCR amplification of the yeast ACT1, HAC1 and SSA4 genes The three yeast genes were amplified in separate PCR reactions using 500 ng of chromosomal yeast DNA as template in PCR buffer (10 mM Tris–HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl2 ), containing 200 ␮M of each dNTP and 10 pmol of each primer in following combinations: (a) ACT1 forward, 5 -NH2 C6-gtattgtcaccaactgggacgatatg-3 and ACT1 reverse, 5 -gaaacacttgtggtgaacgatagatg-3 ; (b) HAC1 forward, 5 -NH2 -C6-aaaagaggaaaaggaacagcga-3 and HAC1 reverse, 5 -catgaagtgatgaagaaatcattca-3 ; and (c) SSA4 forward, 5 -NH2 -C6-cgatagggttgaaattatcgctaacg-3 , and SSA4 reverse, 5 -ctaatcaacctcttcaaccgttggg-3 . The reagents were mixed, and 0.5 units of AmpliTaq Gold polymerase (Applied Biosystems, USA) was added to each PCR tube. The reactions were intitiated by incubation at 95 ◦ C for 7 min, followed by 30 cycles of PCR using a cycle profile of denaturation at 94 ◦ C for 1 min, annealing at 50 ◦ C for 1 min, and extension at 72 ◦ C for 2 min, followed by a final extension of 10 min at 72 ◦ C. One microlitres aliquots of the amplification products were analyzed by electrophoresis in 1% agarose gels. The PCR fragments were purified using QiaQuick PCR purification columns (Qiagen, USA) according to the manufacturer’s instructions, followed by precipitation for 2 h at −20 ◦ C by adding 0.1 vol. of 3 M NaOAc, pH 5.2 and 1 vol. of isopropanol. After centrifugation in a microcentrifuge at full speed for 30 min, the DNA pellets were washed with 70% EtOH, dried (SpeedVac) and resuspended in 10 ␮L water. The desalted PCR samples were stored at −20 ◦ C, until array printing. The corresponding yeast gene fragments without a 5 amino modification were generated by a second PCR using the purified PCR samples as templates and the reverse primers from above combined with the following forward primers: (a) ACT1 forward, 5 -gactcctacgttggtgatgaagctc-3 ; (b) HAC1 forward, 5 -tggaaatgactgattttgaactaactag-3 ; and (c) SSA4 forward, 5 -gctgttggtattgatttaggtacaacc-3 .

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2.3. Oligonucleotide capture probe design and microarray fabrication All capture probes were 70-mer DNA oligonucleotides synthesized with a 5 -NH2 -C6 linker for covalent attachment onto the Immobilizer-coated polymer slide (Exiqon, Denmark). Besides the Yeast Genome Sample Oligo Set (Operon (Qiagen), USA), 13 oligonucleotides were selected from the Mouse Genome Sample Oligo Set (Operon (Qiagen), USA) and used as negative controls. Nine additional 70mer oligonucleotides were designed towards selected yeast genes, by genome-wide BLAST analysis. The design criteria included minimized cross-hybridization, with the maximum allowed overall sequence identity and contiguous stretch of complementary sequence of 75% and 17 nt, respectively, to non-target gene sequences. Finally, the 70-mer sequences with a Tm close to the average Tm for the Operon Yeast Genome Sample Oligo Set were selected for synthesis. The four 50-mer oligonucleotides (with and without 5 -NH2 -C6 modification) designed for exons 8–11, respectively, in the Caenorhabditis elegans let-2 gene were kindly provided by Dr. Dan Jeffares, Department of Evolutionary Biology, University of Copenhagen, Denmark. The oligonucleotides (10 ␮M in 1× Immobilizer spot buffer, Exiqon, Denmark) were spotted onto Immobilizer slides using a Packard BioChip Arrayer I according to the manufacturer’s instructions (Perkin-Elmer Life Sciences, USA). The oligonucleotides were printed in quadruplicate on the slides alongside with the Cy3-labelled 5 NH2 -C6 linked marker oligonucleotide. After printing, the slides were placed in a humidity chamber containing filter paper pre-wetted in saturated sodium chloride solution for 12 h at room temperature for coupling. Finally, the slides were washed in 1 × SSC and dried by centrifugation in slide racks for 2 min in a swing-bucket rotor with microtiter plate buckets at 170 × g. The 5 -NH2 -C6 modified 70-mer oligonucleotide capture probes for yeast ACT1, HAC1 and SSA4 genes along with the corresponding PCR amplicons were spotted onto Immobilizer-coated polymer slides (Exiqon, Denmark) and 3-D LINK slides (Motorola, USA) according to the manufacturers instructions. Prior to printing, all PCR amplicons were quantified to adjust the final concentration at approximately

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400 ng/␮L. The 70-mer capture probes were diluted to 20 ␮M final concentration and the PCR amplicons at 200 ng/␮L with the 2 × spotting buffer recommended by each manufacturer. The arrays were printed onto the different microarray slide types using the BioChip Arrayer I with a 400 ␮m pitch between spots and 3 × 300 pl/spot. The 3D-Link slides and Immobilizer slides were processed according to the manufacturers instructions. 2.4. RNA extraction and synthesis of fluorochrome-labelled cDNA target Yeast total RNA was extracted using the FastRNA Kit-RED (BIO 101, USA) according to the manufacturer’s instructions. Total RNA was extracted from C. elegans worm samples (see Jacobsen et al., 2004, for details of C. elegans cultivation) using the FastRNA Kit, GREEN (QBiogene, Carlsbad, California, USA). The quantity and quality of the total RNA preparations were assessed by standard spectrophotometry or using a NanoDrop ND-1000 (NanoDrop, USA) combined with agarose gel electrophoresis. Fluorochromelabelled first strand cDNA target was synthesized from 15 ␮g of total yeast RNA or 10 ␮g of worm total RNA combined with 5 ␮g anchored oligo(dT) primer (dT20 VN) and subsequently purified as described (Bowtell and Sambrook, 2002).

2.5. Microarray hybridizations The Cy3- and Cy5-labelled cDNA target samples were combined in a 20 ␮l final volume in hybridization solution containing 3 × SSC; 25 mM HEPES, pH 7.0; 0.3% SDS and 1 ␮g/␮L yeast tRNA. The labelled cDNA target sample was filtered in a 0.22 ␮m spin column (Millipore, USA), heated at 100 ◦ C for 2–5 min, cooled for 2–5 min by spinning at max speed in a microcentrifuge at 25 ◦ C, and applied to the microarrays under a Lifter-Slip (Erie Scientific, USA). The arrays were hybridized in a waterbath, in sealed, watertight hybridization chambers (DieTech, USA) for 16–18 h at 65 ◦ C. After hybridization the slides were rinsed in a coupling jar containing 2 × SSC + 0.1% SDS, followed by washing for 1 min in 1 × SSC, then for 1 min in 0.2 × SSC, and finally for 10 s in 0.05 × SSC. The slides were dried as described above. 2.6. Microarray data analysis The slides were scanned using a Packard ScanArray Express HT laser scanner (Perkin-Elmer Life Sciences, USA) and analysed with the GenePix Pro 4.0 (Axon, USA) software. For the comparative hybridizations, high quality spots were defined as features, where at least 50% of the feature pixels had higher intensity than two standard deviations above the local background in at least one of the two fluorochrome channels. The final data analysis included only high quality

Table 1 Fold increase in the expression of seven yeast HSP genes following a 30-min heat shock treatment Gene

ORF

ID

Fold increase Slide A1

SSA1 HSP26 SSE2 HSP78 HSP78 SSA4 SSA4 HSP12 HSP82

YAL005C YBR072W YBR169C YDR258C YDR258C YER103W YER103W YFL014W YPL240C

Operon Operon Operon Exiqon Operon Exiqon Operon Operon Operon

Slide A2

Slide A3

Average

S.D.

Average

S.D.

Average

S.D.

2.7 17.0 6.3 nd 5.3 16.5 nd 12.3 2.4

0.1 1.9 1.3 nd 1.6 2.1 nd 2.3 0.1

2.1 12.4 3.8 4.9 4.2 13.3 14.1 25.7 nd

0.1 1.8 0.4 1.1 0.5 0.9 7.3 5.1 nd

1.8 15.0 4.4 3.8 4.4 12.3 21.3 14.2 3.8

0.4 3.9 0.6 0.3 0.4 1.2 9.9 2.8 0.2

The calculated expression ratios from each experiment were averaged from four replicas, while nd denotes spots where high quality or positive signals could not be detected for all four replicas. The cDNA derived from heat shocked yeast cells was labelled with Cy5 for slides A2 and A3, and with Cy3 for slide A1 (dye-swap). The two oligonucleotides designed and synthesized in this study are depicted as Exiqon.

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spots. The datasets were normalized in each microarray using global normalization in which the mean ratio of all array elements was equalized to 1. After normalization, expression levels were calculated for each array element as a ratio of local background-subtracted fluorescence intensity values. The coefficient of variation (CV%) is calculated from the standard deviation of the entire population of arguments divided by the average value of the arguments multiplied by 100. For coefficient of variation of spot intensities or fluorescence intensities, the arguments are background-subtracted fluorescence intensities for the individual spots. For the coefficient of variation of ratios, the arguments comprise the ratio of means for all high quality spots included in Table 1.

3. Results and discussion In the fabrication of pre-spotted microarrays where liquid droplets are robotically deposited on the array support, it is important to know how the liquid spreads out over the slide surface. The resulting droplet size is related to the hydrophobicity of the surface as measured by the contact angle. A low contact angle (<45◦ ) indicates a hydrophilic surface with good wetting properties on which a droplet will readily spread and stick, while a high contact angle (>90◦ ) indicates a hydrophobic surface where the droplets do not stick, but can be easily displaced. Whereas a small spot size is a prerequisite for the fabrication of high-density microarrays, firm attachment to the underlying support is also needed so that the capture probe droplets remain in position where they can dry out to produce uniform high-quality microarrays. One of the key features of the anthraquinone-based immobilization technique (Koch et al., 2000) is the ability to efficiently coat a raw polymer surface by UV-light induced binding of the photoreactive Immobilizer reagent. Coating of the injection-moulded polymer microarray slide with the reagent results in a decrease in hydrophobicity of the surface, thus, enabling fabrication of high-density microarrays with a reproducible spot size. This was assessed by fabricating a grid of 40 spots of a Cy3labelled, 5 -NH2 -C6-modifed oligonucleotide using a 300 pl spotting volume, which resulted in relatively small (80 ␮m), homogenous spots on the Immobilizer-

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coated polymer slide surface (Fig. 1A) with a spot fluorescence intensity CV% of 6.2. By comparison, the same Cy3-labelled oligo droplets spotted on a coated glass microarray support (Fig. 1B) were significantly larger (220 ␮m) and more irregular due to the lower contact angle resulting in a higher fluorescence intensity CV% of 28 (for the specification of CV% calculation see Section 2). Autofluorescence of the coated polymer slide was investigated using standard laser and photo-multiplier (PMT) settings (80% laser power, 80% PMT) on a confocal laser scanner. An average autofluorescence of 225.8 and 297.2 was measured for Cy5 and Cy3, respectively, across the printing window on three slides (data not shown), indicating that autofluorescence in the polymer slide is sufficiently low to allow expression profiling by hybridization to spotted microarrays. The slide-to-slide variation of the Immobilizer-coated polymer slides was addressed by fabricating arrays of the same Cy3-labelled, 5 -NH2 -C6-modifed oligonucleotide onto eight polymer slides using a spotting volume of 3 × 300 pl, with each array comprising 40 spots. Scanning of the resulting arrays revealed an average fluorescence intensity of 6639.4 with a CV% of 12.1 across the eight slides (Fig. 1C). Combined with the results from Fig. 1A, this indicates that the slide surface is highly homogeneous both within a single slide as well as between different slides. The uniform layer of electrophilic groups introduced onto the coated polymer slide by photocoating with the Immobilizer reagent enables a one-step coupling of spotted 5 amino-linked oligonucleotides onto the polymer slide. To assess non-specific binding, we compared the average hybridization signals obtained from four different 50-mer oligonucleotide probes synthesized with or without a 5 amino-C6 linker. The probes were designed to detect exons 8–11 in the let-2 gene encoding type IV collagen in C. elegans. Hybridization with fluorochrome-labelled cDNA target derived from C. elegans total RNA resulted in five-fold higher hybridization signals from the 5 amino-modified oligonucleotide capture probes compared to those obtained with unmodified 50-mer probes (Fig. 1D). This implies that binding of spotted oligonucleotides onto the slide surface via exocyclic amine groups is low. To directly compare the performance of the Immobilizer polymer slide with a commercially available

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Fig. 1. Comparison of coated polymer and glass microarray slide surfaces. Scanned images of 300 pl droplets of a Cy3-labelled NH2 -C6modified oligonucleotide deposited on the (A) Immobilizer-coated polymer and (B) pre-activated 3D-Link glass slide, respectively, using the BioChip Arrayer I non-contact spotter from Packard. (C) Average Cy3-fluorescence intensities for 40 spots of a Cy3-labelled, 5 -NH2 -C6modified oligonucleotide spotted on eight slides from a single slide production batch. The spotting volume was 900 pl. (D) Average fluorescence intensities obtained from two-colour hybridizations to four 50-mer capture probes designed for the C. elegans let-2 gene, synthesized with or without 5 -NH2 -C6-modification. The data shown are the mean ± S.D. in each case.

glass microarray slide, the pre-activated 3D-Link slide from Motorola, we selected a small subset of yeast capture probes for monitoring expression of the yeast ACT1, HAC1 and SSA4 genes. Of these, the ACT1 gene encoding yeast actin has 21 mRNA copies per yeast cell, the HAC1 gene encoding a bZIP transcription factor has 4 copies per yeast cell, while the SSA4 gene encoding the heat shock protein HSP70 has 1 mRNA copy per yeast cell at standard growth conditions (Causton et al., 2001; Gasch et al., 2000). Furthermore, expression of SSA4 is induced by heat shock, while expression of HAC1 is slightly repressed and expression of ACT1 is unaffected by heat treatment. The capture probes were synthesized both as aminolinked PCR amplicons and 70-mer oligonucleotides directed towards the 3 -end of each open reading frame. The 5 amino-modified capture probes were used both for the Immobilizer slide and the 3D-Link glass slide. The 70-mer oligos and PCR amplicons were spotted

onto the microarray slides in quadruplicate and used to compare gene expression in standard grown and heat-shocked wild type yeast (Fig. 2). Two-colour hybridizations using the same Cy3- and Cy5-labelled cDNA derived from the two yeast total RNA samples revealed a heat-shock induced expression of the SSA4 gene both by 70-mer oligos and PCR amplicons spotted onto the Immobilizer and 3D-Link slides, respectively (Fig. 2A and B). By comparison, expression of the ACT1 gene was, as expected, not affected by heat stress, whereas HAC1 expression was slightly down regulated (Fig. 2A and B). Furthermore, the calculated fold change for SSA4 expression was 4 (70mer oligo) to 8 (PCR amplicon) on the Immobilizer slide array, whereas a lower fold change of 3 (70-mer oligo) to 1.5 (PCR amplicon) was found for the 3DLink array. Nevertheless, both slide types were able to identify the heat stress induced expression of the SSA4 gene.

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Fig. 2. Direct comparison of two different microarray slide surfaces in measuring gene expression of three yeast genes. The PCR amplicons and 70-mer oligonucleotide capture probes for the yeast ACT1, HAC1 and SSA4 genes were spotted in quadruplicate onto the (A) Immobilizer-coated polymer slide and (B) 3D-Link slide from Motorola. The spotted arrays were hybridized with the same Cy3- and Cy5-labelled cDNA target pool, reverse transcribed from standard-grown (green) and heat shocked (red) wild type yeast total RNA, respectively, for which the scanned images are shown. The average fluorescence intensities for the 70-mer oligos and PCR amplicons are shown below for the three yeast genes.

To demonstrate the utility of our coated polymer slide in gene expression profiling, we produced spotted oligonucleotide microarrays using a collection of 5 amino-modified 70-mer oligonucleotide probes representing 96 yeast genes (Operon Yeast Genome Sample Oligo Set) and 13 mouse genes as negative con-

trols (Operon Mouse Sample Oligo Set). In addition, we designed and synthesized 70-mer oligonucleotide probes for nine selected yeast genes represented in the Operon Yeast Set that were spotted alongside the Operon probes. The oligonucleotide capture probes were spotted in quadruplicate and used to compare

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Table 2 Fold increase in the expression of the SSA4 gene following a 30-min heat shock Gene

ORF

ID

Wild type/∆SSA4 ratio Slide B1

SSA4 SSA4

YER103W YER103W

Exiqon Operon

Slide B2

Slide B3

Average

n

Average

n

Average

n

42.2 31.3

2 3

48.3 40.2

3 4

55.5 74.1

4 4

Fold increase is derived from comparison between heat-shocked wild type and SSA4 deletion mutant yeast cells. The cDNA derived from wild type yeast cells was labelled with Cy3 for slides B1 and B2, and with Cy5 for slide B3 (dye-swap). The negative signals in four out of the 24 replicas were excluded (see text for detailed description) and average ratios were calculated from the remaining two to four replicas.

gene expression in two total RNA samples extracted from standard grown and heat-shocked wild type yeast cells, respectively. Two-colour hybridizations were carried out using Cy3- and Cy5-labelled cDNA target pools derived from the two yeast total RNA samples. Three independent hybridizations were performed, one of which (slide A1, Table 1) was a dye-swap experiment, and the gene expression ratios were calculated for each element on each microarray by including only the probes, where all four replica spots gave rise to a high quality hybridization signal. In the comparison of reference versus heat-shocked wild type yeast cells, the 70-mer oligonucleotide microarrays could reproducibly measure heat stress induced expression of seven yeast heat shock protein (HSP) genes represented in the Operon Yeast Set (Table 1). Furthermore, the results in Table 1 demonstrate the flexibility of the polymer microarray platform, i.e., the oligonucleotide capture probes designed and produced in our laboratory for two heat shock genes (YDR258C/HSP78 and YER103W/SSA4) perform equally well as those from the Operon Yeast Set. Next, we evaluated the specificity of the polymer microarray platform by comparing total RNA samples extracted from heat-shocked wild type yeast and a strain deleted for the SSA4 gene, ∆SSA4. This allowed us to measure the signal-to-noise ratios for the SSA4-specific oligonucleotide probes corresponding to the fluorescence intensity detected from the wild type yeast RNA relative to that from the ∆SSA4 strain. Twocolour hybridizations were carried out using Cy3- and Cy5-labelled cDNA targets reverse transcribed from the two yeast total RNA samples, and three replicate hybridizations were performed, including a dye-swap experiment (slide B3, Table 2). Subsequent calculations of the signal-to-noise ratios for each SSA4 cap-

ture probe on each microarray (Table 2) revealed an average ratio ranging from 31- to 74-fold between the two yeast strains. The fact that the SSA4 capture probes in some cases showed fluorescence intensities, which were lower than that of the local background when hybridized with the ∆SSA4 cDNA target, thus resulting in negative signals in four out of the 24 replicas analysed, clearly demonstrates the very low level of cross-hybridization from adequately designed oligonucleotide probes spotted on the polymer microarray platform. Given the importance of optimal probe selection (Li and Stormo, 2001) and quality, we were pleased to find good overall agreement between the results obtained with the Operon SSA4 oligonucleotide probe and the 70-mer probe designed and produced in this study to detect the SSA4 mRNA in yeast (Table 2 and Fig. 3). Genome-wide expression analysis of the heat shock response in S. cerevisiae has previously been interrogated using spotted cDNA microarrays (Gasch et al., 2000) and high-density 25-mer oligonucleotide arrays from Affymetrix (Causton et al., 2001). The expression data of seven yeast heat shock genes from the present report and the two published studies are compared in Table 3. Although limited, our data obtained using 70mer oligonucleotide probes spotted onto the polymer slide platform show comparable results to those reported by the two previous studies, i.e. within a two- to three-fold difference in the fold increase measured with the different microarray types (Table 3). Two yeast heat shock genes, HSP12 and HSP26, showed discordant expression data compared to the Affymetrix GeneChip study (Causton et al., 2001), which reported a significantly higher (>200) fold increase in expression, as opposed to those detected here and by Gasch et al. (2000). Poor correlation between expression data obtained from Affymetrix GeneChips and spotted cDNA

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Table 3 Comparison of the average fold increase in the expression of seven HSP genes in three yeast expression profiling studies Average fold of induction Gene

ORF

This study

cDNA arraya

Affymetrixb

SSA1 HSP26 SSE2 HSP78 SSA4 HSP12 HSP82

YAL005C YBR072W YBR169C YDR258C YER103W YFL014W YPL240C

2 15 5 5 15 17 6

1 6 8 11 18 62 4

4 221 12 5 19 213 3

a b

Gasch et al. (2000). Causton et al. (2001).

microarrays has previously been reported by Kothapalli et al. (2002) and Kuo et al. (2002). While both studies concluded that the discrepancies could be due to the lack of sequence specificity of spotted cDNA arrays

Fig. 3. Comparison of heat shock response in wild type yeast and a SSA4 deletion strain. Scanned image of a two-colour hybridization of the yeast 70-mer oligonucleotide microarray with Cy5- and Cy3labelled cDNA, reverse transcribed from heat shocked wild type and ∆SSA4 yeast total RNA. The spots representing SSA4-specific 70mer oligonucleotides from either Operon or Exiqon are indicated below in the enlarged image. In this microarray experiment only spots representing the SSA4 gene are expected to show up in red. The calculated signal-to-noise ratios for the SSA4 gene in heat shocked wild type versus ∆SSA4 yeast strains, respectively, are shown in Table 2.

as well as errors in tracking of PCR amplified cDNA fragments, the dissimilarities found in the latter study involved expression data sets derived from two different laboratories using different materials and protocols (Kuo et al., 2002). Thus, the observed discrepancies in the expression of the two HSP genes reported here can at least partly be explained by the use of different yeast strains, growth conditions and different temperature regimes during the heat-shock treatment. For example, in their genome-wide expression profiling of heat-shock responses in yeast, Gasch et al. (2000) used two heat stress time courses and observed significant variation between the two expression data sets. In order to assess the variance in our polymer microarray platform, we carried out three independent two-colour hybridizations, including a dye-swap, for each microarray experiment, using the same batch of spotted 70-mer oligonucleotide microarrays, but different sets of yeast cells and thus, different total RNA samples. Given the observed average coefficient of variation of 25.9% on the ratios (Table 1) across repeated hybridizations, we believe that our polymer microarray platform is of adequate quality generating reliable and reproducible data from replicate experiments. In summary, we have shown that 70-mer oligonucleotide microarrays spotted onto coated polymer slides provide biologically valid gene expression measurements comparable to those detected using spotted cDNA arrays and short oligonucleotide arrays from Affymetrix. This is in good agreement with two recent studies reporting a strong correlation between spotted unmodified 70-mer microarrays and cDNA arrays (Wang et al., 2003) and spotted 5 amino-modified oligonucleotide arrays and Affymetrix GeneChip ar-

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rays (Barczak et al.,2003). Taken together, our findings strongly suggest that the coated polymer microarray slide described here represents a robust and costeffective array platform for pre-spotted oligonucleotide arrays, capable of producing accurate, reproducible and valid data.

Acknowledgements Excellent technical assistance from Anja Konge, Janni J. Jørgensen, Kirsten Gerner-Smidt, Mette R. Larsen and Morten Bruus is gratefully acknowledged.

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