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Technical support : RNA Amplification
The direct conversion of mRNA into labeled cDNA requires substantial amounts of template that are not always available. This is the case when using microdissected tissues or small populations of rare cells isolated by FACS. In such cases, mRNA should be amplified to an amount sufficient for synthesis of microgram quantities of target necessary to enable efficient array detection. The most commonly used method is referred to as the antisense RNA (aRNA) amplification procedure developed in the lab of J. Eberwine (Van Gelder et al., PNAS, 87 : 1663). When compared to other methods, this amplification procedure for gene expression studies has the advantage not to significantly distort the relative abundance of individual mRNA species within a RNA population.
The procedure consists of reverse transcription with an oligo(dT) primer bearing a T7 promoter and in vitro transcription of the resulting DNA with T7 RNA polymerase to generate appreciable amounts of aRNA from an initial RNA input as little as 2 µg of total cellular RNA or 1 ng of mRNA (see Figure). The aRNA unlabeled products can then be processed using reverse transcription-based labeling method (direct or indirect) to yield labeled cDNA targets for hybridisation onto microarrays with spotted cDNA or antisense oligonucleotide probes. The use of arrays with spotted sense oligonucleotide probes requires the generation of labeled aRNA that can be used directly for hybridisation. Commercial RNA amplification kits are available for both strategies (RiboAmp RNA amplification kit, Arcturus, cat# KIT02201; AminoAllyl MessageAmp aRNA kit, Ambion, cat# 1752).
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