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Use of Transcriptor Reverse Transcriptase,,,in Microarray Analysis

ethanol precipitation. The RNA was vacuum dried and resuspended in RNase-free water to a final concentration of 1 g/l.

Target labelling reaction

Labelling was performed using a modified version of the published protocol from the DeRisi laboratory (www.microarrays.org). The modifications included an increase in the ratio of amino-allyl-dUTP:dTTP to 6:4. The neutralisation was performed with Tris-HCl. All reverse transcription reactions were performed at 42C using 15 g of total RNA previously extracted as described above. The total unit amount of Transcriptor Reverse Transcriptase was 20 units per reaction (10 units added, and 1 hour later another 10 units were added). The total unit amount of product Y was 400 units per reaction (400 units added and left for 2 hours according to the manufacturers recommended protocol). The labelled product was vacuum dried and finally resuspended in 34.5 l HGMP hybridisation buffer (40% formamide, 5x Denhardts solution, 5x SSC, 0.05 M Tris-HCl pH 7.4, 1.0 mM sodium pyrophosphate, 0.1% SDS), 0.4 g yeast tRNA, 0.96 g poly-dA and 2.7% SDS (www.hgmp.mrc.ac.uk/Research/Microarray/ index.jsp).

Hybridisation

The microarrays used in this study are from a single printing run, custom made with approximately 12,000 sequenced expressed-sequence tags (ESTs) from a nonnormalised library. The ESTs were PCR amplified and the products used for printing were analysed for content and quality on agarose gels. Every microarray was visually inspected and samples were prescanned for printing defects. The microarrays were preblocked (10% BSA, 3x SSC) for 1 hour at 55C. Slides were washed with Milli-Q water (Millipore), and dried by centrifugation. Fixation of cDNA products was achieved after boiling the product and submersing
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