Validation of a novel, fully integrated and flexible microarray benchtop facility for gene expression profiling

Nucleic Acids Research, 2003, Vol. 31, No. 23 e151 DOI: 10.1093/nar/gng151 Validation of a novel, fully integrated and ¯exible microarray benchtop facility for gene expression pro®ling Michael Baum*, Simone Bielau, Nicole Rittner, Kathrin Schmid, Kathrin Eggelbusch, Michael Dahms, Andrea Schlauersbach, Harald Tahedl, Markus Beier, Ramon GuÈimil, Matthias Schef¯er, Carsten Hermann, JoÈrg-Michael Funk1, Anke Wixmerten, Hans Rebscher, Matthias HoÈnig, Claas Andreae, Daniel BuÈchner, Erich Moschel, Andreas Glathe, Evelyn JaÈger, Marc Thom, Andreas Greil, Felix Bestvater, Frank Obermeier, Josef Burgmaier, Klaus Thome, Sigrid Weichert, Silke Hein, Tim Binnewies, Volker Foitzik, Manfred MuÈller, Cord Friedrich StaÈhler and Peer Friedrich StaÈhler Received July 6, 2003; Revised September 20, 2003; Accepted October 9, 2003 ABSTRACT INTRODUCTION Here we describe a novel microarray platform that integrates all functions needed to perform any array-based experiment in a compact instrument on the researcher's laboratory benchtop. Oligonucleotide probes are synthesized in situ via a lightactivated process within the channels of a threedimensional micro¯uidic reaction carrier. Arrays can be designed and produced within hours according to the user's requirements. They are processed in a fully automatic work¯ow. We have characterized this new platform with regard to dynamic range, discrimination power, reproducibility and accuracy of biological results. The instrument detects sample RNAs present at a frequency of 1:100 000. Detection is quantitative over more than two orders of magnitude. Experiments on four identical arrays with 6398 features each revealed a mean coef®cient of variation (CV) value of 0.09 for the 6398 unprocessed raw intensities indicating high reproducibility. In a more elaborate experiment targeting 1125 yeast genes from an unbiased selection, a mean CV of 0.11 on the fold change level was found. Analyzing the transcriptional response of yeast to osmotic shock, we found that biological data acquired on our platform are in good agreement with data from Affymetrix GeneChips, quantitative real-time PCR andÐalbeit somewhat less clearlyÐto data from spotted cDNA arrays obtained from the literature. Microarrays have become a standard tool in molecular biology that has revolutionized genomics research. Microarrays are used extensively for gene expression pro®ling (1,2) in many applications including the discovery of gene function (3,4), drug evaluation (4±6), pathway dissection (7), classi®cation of clinical samples (8±10), exon mapping (11) and investigation of splicing events (12). Arrays may be produced either by deposition of presynthesized material (1,13±15) or by in situ oligonucleotide synthesis (16,17). DNA arrays manufactured by physical deposition of presynthesized material require labor-intensive preparation and record-keeping of DNA probes. In contrast, oligonucleotide arrays synthesized in situ using a photolithographic method (18) only require DNA sequence data. However, cost and time spent in generating the photolithographic masks render this approach as slow and in¯exible as the deposition methods. Recently, more ¯exible microarray technologies have been developed. These employ either ink-jet printing (19) or micromirror devices (20,21) for in situ synthesis of customized oligonucleotide arrays. Although these techniques provide full ¯exibility with respect to the array design, the actual generation of the array and in some cases even the hybridization and detection steps are restricted to centralized manufacturer facilities. Again, the investigator's ¯exibility remains limited. In addition, array synthesis and subsequent processing steps are not physically linked and require error-prone manual handling. The geniom platform described here is the ®rst system to overcome these restrictions. The investigator gains full control of the complete work¯ow of any microarray experiment. The technology integrates microarray production, hybridization and detection in a compact benchtop unit. Automation of these processes *To whom correspondence should be addressed. Tel: +49 621 3804 257; Fax: +49 621 3804 400; Email: Nucleic Acids Research, Vol. 31 No. 23 ã Oxford University Press 2003; all rights reserved febit ag, KaÈfertaler Strasse 190, 68167 Mannheim, Germany and 1Carl Zeiss Jena GmbH, Carl Zeiss Group, Business Group: Microscopy, Division: Advanced Imaging Microscopy, Carl-Zeiss-Promenade 10, 07745 Jena, Germany e151 Nucleic Acids Research, 2003, Vol. 31, No. 23 MATERIALS AND METHODS Oligonucleotide arrays Light-activated in situ oligonucleotide synthesis was performed essentially as described by Singh-Gasson et al. (20) using a digital micromirror device (Texas Instruments). The synthesis was performed within the geniom device on an activated three-dimensional reaction carrier consisting of a glass-silicon-glass sandwich (DNA processor; see Supplementary Material Fig. 1). Four individually accessible microchannels (referred to as arrays) etched into the silicon layer of the DNA processor are connected to the micro¯uidic system of the geniom device. Using standard DNA synthesis reagents (Proligo) and 3¢-phosphoramidites carrying a 5¢photolabile protective group (22,23), oligonucleotides were synthesized in parallel in all four translucent arrays of one reaction carrier. Prior to synthesis, the glass surface was activated by coating with a spacer. The synthesized probe sets may be the same or different for all four arrays. Actually, the time needed for synthesis of standard arrays used in this study is independent of the number of different probe sets, the probe sequences and the number of probes synthesized within one probe set (current limit: 14 000 features per array; corresponding to 4 3 14 000 = 56 000 features per reaction carrier). However, the probe length substantially in¯uences synthesis time. According to the conservative protocol used in this study, the synthesis of four typical 25mer arrays (with 12 880 features each) takes ~15.5 h (including 1.5 h for the ®nal deprotection step). The yeast probe set (ten 25mer probes per transcript) was calculated based on the full genome sequence (retrieved online from http://genome-www.stanford.edu/ Saccharomyces/) using a combination of sequence uniqueness criteria and rules for selection of oligonucleotides likely to hybridize with high speci®city and sensitivity. The selection criteria were essentially as described in Lockhart et al. (2) with modi®cations for the longer probes used here (25mers instead of 20mers). Yeast strain and growth conditions Saccharomyces cerevisiae, wild-type strain W303-1A, MATa, ura3-52, trp1D2, leu2-3_112, his3-11, ade2-1, can1-100 (accession no. 20000A; EUROSCARF, Frankfurt a.M., Germany) was grown in 240 ml batch cultures at 30°C in YPD (1% yeast extract, 2% peptone, 2% glucose) to an A600 of 1.0. At this point, cells were collected for determination of expression p (...truncated)