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Use of Transcriptor Reverse Transcriptase,,,in Microarray Analysis

nitor gene expression in both host and pathogen [1]. However, the system must be optimised for pathogen gene expression since the T. gondii RNA can be more than an order of magnitude less concentrated compared with the host cell RNA. Over the past few years, technological advances have made the method much more reliable and robust.

Curiously, the standard protocols for generating the labelled cDNA target from the experimental RNA, arguably the most critical step in the procedure, remain virtually unchanged. The vast majority of investigators use either a direct incorporation of deoxynucleotides with covalently attached fluorophores or the incorporation of amino-allyl deoxynucleotides with subsequent fluorophore conjugation. In both methods, the efficiency of the target labelling reaction and quality of the labelled cDNA product depend almost entirely on the reverse transcription enzyme used.

We employ an amino-allyl labelling reaction to generate and compare microarray data from different commercially available reverse transcriptases.

Materials and Methods

Parasite and RNA preparation

Parasites (RH strain) were cultured according to a standard protocol. NIH 3T3 cells were used as host cells, and the parasites were harvested by centrifugation just prior to monolayer lysis. The cells were suspended in TRI reagent (Sigma). RNA was extracted according to the manufacturers protocol, except that the RNA was not ethanol precipitated following incubation with 2-propanol, but was added directly to an RNeasy Midi column (Qiagen). Binding of the sample was followed directly with the recommended wash and two extra washes. The RNA was eluted with a total of 450 l RNase-free water. This was followed by a standard
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