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Automated RNA Amplification for Arrays

agents for the cDNA synthesis and amplification steps. Purification was conducted with magnetic beads for capture and elution of cDNA and aRNA.

Figure 1. Average Yield and Purity of aRNA Synthesized on a High Throughput Platform. (A) Eight 2 g replicates of three different total RNA samples (Ambion #7836 HL60 Promyelocytic Leukemia, Ambion #7960 Human Liver, Stratagene #740000 Universal Human Reference total RNA) were amplified and purified on two plates, for a total of 48 different samples. Samples were prepared as described by the Amino Allyl MessageAmp aRNA Kit protocol modified for robotic distribution of reagents for the cDNA synthesis and amplification steps. Purification was conducted with magnetic beads for capture and elution of cDNA and aRNA. (B) Yield and purity (A260:A280 absorbance ratio) were measured on a Tecan Genios Multi-Detection Microplate Reader using a 384 well BD Falcon Microtest UV-Vis microplate.


Similar results were also obtained using 1 g of starting material and typical yields from 1 g of the four different total RNA samples (brain, liver, HL60, and Universal Human Reference RNA) averaged 2040 g of aRNA, with purity equivalent to that obtained with 2 g. These results suggest that this automated process consistently yields a 500-fold or greater increase in aRNA product from total RNA sample.

Samples prepared by automation were evaluated by hybridization on a BGA plate, with pre- and post-hybridization processing performed with a robotic liquid handler. Figure 2 demonstrates representative
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