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Use of Transcriptor Reverse Transcriptase,,,in Microarray Analysis

ource is used throughout and each target labelling represents an independent reverse-transcriptase and dyecoupling reaction. Data from each microarray are derived from two independently labelled targets simultaneously hybridised to a single microarray.



Although the relative specific activities of a hybridised labelled target can be estimated by total mean fluorescence over an entire microarray signal set, the distribution of signal intensities on a per-spot (probe) basis is more informative. Figure 2 illustrates a histogram of three representative sets of 9,737 raw (nonnormalised) fluorescent signals from the Cy5 channel. Compared with Transcriptor Reverse Transcriptase there is a much greater abundance of signals in the 0 5,000 class and relative paucity of signals in the higher fluorescence classes for product X. This signal distribution indicates that Transcriptor Reverse Transcriptase labels cDNA more efficiently than product X. In contrast, Transcriptor Reverse Transcriptase and product Y show similar signal distributions where more than half of the signals were in classes greater than 5,000. Members of the highest signal class are signals at saturation, some of which are T. gondii gene transcripts known to be in very high abundance. The high number of signals in this class for Transcriptor Reverse Transcriptase indicates a relatively strong labelling reaction compared with the low signal numbers for product X. Since the optimal laser power and PMT levels were similar for all three microarray scans, image acquisition can not account for the difference in results.



Consistency of target labelling is critical for microarray analysis of gene expression to detect real transcriptional differences between experimental treatments. Since the same input
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