( ...)Use of Transcriptor Reverse Transcriptase,,,in Microarray Analysis

Researchers studying tissue and cellular differences in gene expression have applied microarray analysis to questions ranging from cancer research to plant development studies. Although the method is well established, many of the steps in the process continue to be optimised to increase overall sensitivity and reliability. Here, we monitor gene expression in the obligate intracellular pathogen Toxoplasma gondii in order to compare reverse transcriptase target labelling performance on a custom T. gondii 12,000 cDNA microarray. A histogram of signal intensities shows that the Transcriptor Reverse Transcriptase produced a major shift in the signal distribution to higher signal intensity classes compared with the most commonly used RNase H- M-MuLV reverse transcriptase. This indicates the generation of a labelled target with an overall higher specific activity. The correlation of Cy5/Cy3 ratio data from four replicate microarrays demonstrate that target labelling with Transcriptor Reverse Transcriptase is highly reproducible and produces fewer outlier signals compared with the updated version of the RNase H- M-MuLV reverse transcriptase. The known enzymatic properties of Transcriptor Reverse Transcriptase suggest that it will produce a labelled target that closely reflects the experimental input RNA.

Introduction

Microarray analysis of gene expression is rapidly becoming a common tool for molecular biological investigations. The ability to simultaneously monitor tens of thousands of genes affords an unprecedented view of the transcriptional changes that underlie cellular processes. Microarrays are ideal for genetic investigations of intracellular pathogens such as Toxoplasma gondii because it is possible to simultaneously mo
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