( it in 95% ethanol. It was prehybridise...)Use of Transcriptor Reverse Transcriptase,,,in Microarray Analysis

it in 95% ethanol. It was prehybridised (0.001% SDS, 10% BSA, 3x SSC) at 55C for 25 minutes, washed twice in Milli-Q water and washed in 100% ethanol. Hybridisation was carried out in a humid Arraylt hybridisation cassette using a lifter slip coverslip (Erie Scientific) overnight in a 42C water bath. Post hybridisation, the slides were washed for 4 minutes in progressively reduced SDS concentrations (solution 1: 0.5x SSC, 0.1% SDS; solution 2: 0.5 x SSC, 0.01% SDS; solution 3: 0.06 x SSC). Finally, the slides were dried by centrifugation.

Spot finding and scanning

The slides were scanned using an Axon 4000A Scanner according to the manufacturers instructions. The images were spot-found using GenePix 4.1 software with a fixed spot diameter of 150 mm. The median fluorescence value for the spot was used for final analysis without background correction or within chip normalisation.

Data analysis

Microsoft Excel was used to create the correlation curves and histogram data. The Cy5/Cy3 data were also exported from Microsoft Excel into GeneSpring 6.0 for final analysis of gene-specific effects.

Results and Discussion

Since a single microarray experiment consists of multiple steps, the evaluation of any particular step is assessed from the resulting data. Ideally, the labelled target should accurately reflect the input mRNA in both content and representation, have a high specific activity to maximise sensitivity and be very consistent between reactions using the same input mRNA. The first of these properties is inferred from prior enzymology. The second and third properties are experimentally evaluated using the scheme outlined in Figure 1. Briefly, a single RNA s
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