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Is RNA Amplification Necessary for Microarrays?


RNA amplification using the Van Gelder and Eberwine technique (1) has been used extensively for target synthesis and labeling in array analysis experiments. Amplification of RNA is clearly necessary when working with limited samples (needle biopsies, LCM samples, etc.), but is it necessary when the availability of sample is not limiting? Is there additional biologically relevant information that can be obtained from array analysis using amplified RNA as a target? In this article we provide a brief synopsis of the findings of Polacek et al. (2) and Feldman et al. (3) on why amplified RNA (aRNA) might be a better choice than unamplified RNA for microarrays.


The Polacek Experiments
Polacek's group tested the fidelity of differential gene expression data using amplified RNA in a well established in vitro model of cytokine [tumor necrosis factor (TNF-α)]-stimulated human aortic endothelial cells (HAEC). 100 ng of total RNA, isolated from HAECs using a glass filter based kit, was amplified using Ambion's MessageAmp aRNA Kit, producing 4-5 g of aRNA (cRNA). 2 g of this aRNA was labeled with 33P by reverse transcription using hexanucleotide random primers; 10 g of Total RNA (unamplified) was labeled with 33P by reverse transcription using Oligo d(T)15 primers. These targets were hybridized to arrays containing
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