cDNA clone inserts (not sequence-verified) from eighty-eight 384-well microtitre plates from the Human Unigene 1 clone set (RZPD Deutsches Ressourcenzentrum für Genomforschung and Boer et al. 2001) and a custom-made plate of an additional 258 clones (sequence-verified) were amplified by PCR in a 384-well format using M13 forward (5`-CGTTGTAAAACGACGGCCAGT-3´) and reverse (5´-TTTCACACAGGAAACAGCTATGAC-3´) primers. The PCR reaction was set up by dipping a 384-pin replicator (Genetix, UK) twice into the bacterial library plate and transferring the solution (approximately 0.2 µL) to a new PCR plate containing 25µL of PCR master mix in each well (final concentrations: 1 X PCR buffer (0.5M Tris-HCl (pH 8.6), 0.5M KCl, 15mM MgCl2, 1% Tween 20), betaine (1.5U), 0.2mM dNTPs, 0.2mM primers and 0.1U/µL Taq/Pfu). Amplification of PCR products was carried out as follows: 95ºC for 1 min, 94ºC for 15 sec, 59ºC for 15 sec, 65ºC for 55 sec, repeat of 59ºC for 15 sec, 65ºC for 55 sec for an additional 2 cycles, repeat of 94ºC for 15 sec, 59ºC for 15 sec, 65ºC for 55 sec for 29 cycles and finally 68ºC for 10 min. The sub-cycling step described improved product yield and stability. The quality of the PCR reaction was determined visually by slot-blot electrophoresis on 1% agarose gels.
Prior to spotting, PCR products were denatured by the addition of a final concentration of 0.1M NaOH to each well. The ~34,000 PCR products were repeatedly spotted 7 times in a 6 X 6 pattern (asymmetrically to ensure ease in determining filter orientation) on dry 23 X 23 cm Hybond N+ nylon membranes (Amersham Pharmacia) using a Genetix robot attached to a 384 pin gadget-head (250 mm pins). The 6 X 6 spotting blocks were organized into six fields of 384 spotting blocks (arranged 16 X 24), resulting in 2304 spotting blocks on each filter. Each 6 X 6 spotting block contained duplicate PCR products from 15 clones (14 clones in field 6), duplicate Arabidoposis thaliana clones (GenBank accession numbers AF104328 and U29785, derived from the Arabidopsis Biological Resource Center and DNA stock donor at Ohio State University) serve as guide spots during subsequent gridding steps, and four (six in field 6) empty positions to allow for an estimation of background intensity after hybridization. In order to determine the quality of the spotting, 1.58 µM fluorescin was added to the Arabidoposis thaliana control clone plate and the filter was scanned using an ‘in-house’ work station for high resolution fluorescence detection (MPI for Molecular Genetics, Berlin). In this way, unevenly or poorly spotted filters were discarded prior to hybridization. After spotting, cDNA on the filters was further denatured by floating filters (face up) in 0.4M NaOH for 2 min and neutralized with 5 X SSC (pH 7.5) for 2 min. The DNA was cross-linked to the filter by heat-baking at 80°C for 30 min and exposing to ultraviolet radiation (Stratalinker UV cross-linker (auto cross-link option); Stratagene). Filters were stored dry at 4°C until ready for use.
Reference: Boer, J.M., Huber, W.K., Sultmann, H., Wilmer, F., von Heydebreck, A., Haas, S., Korn, B., Gunawan, B., Vente, A., Fuzesi, L. et al. (2001) Identification and classification of differentially expressed genes in renal cell carcinoma by expression profiling on a global human 31,500- element cDNA array. Genome Res, 11, 1861-1870.