RNAi for High Throughput Analysis

Large scale gene expression studies require reliable high-throughput methods to synthesize and then purify high quality RNA from a large number of samples. Although the MEGAscript RNAi Kit includes reagents for both dsRNA production and clean up, it is not optimized for clean up in a 96 well plate format. Thus for high-throughput applications, we recommend combining Ambion's MEGAscript High Yield Transcription Kit with MEGAclear-96, which is designed specifically for high-throughput and rapid clean up of large scale transcription reactions.

To demonstrate the utility of this approach, Ambion developed an automation protocol based on the MEGAclear^™ technology to purify RNA from high yield in vitro transcription reactions. Two dsRNAs specific for the U2AF⁵⁰ gene product, synthesized using MEGAscript, were purified using the MEGAclear^™-96 Kit on the Packard MultiPROBE II HT or the Biomek 2000 platforms. The activities of the purified dsRNAs were then tested in Drosophila L2 cells. As shown in Figure 1, a silencing effect similar to dsRNAs prepared and purified with the MEGAscript^™ RNAi Kit was observed.

The MEGAclear-96 Kit can clean up as many as 96 samples in just half an hour. This procedure can be integrated with your favorite robotic platform or used with manual methods for many downstream applications, including RNA interference, aRNA amplification, array hybridization and translation studies.

Figure 1. Comparison of dsRNA Prepared and Purified with the MEGAscript™ RNAi Kit vs. the MEGAscript Kit Followed by MEGAclear™-96. Western blot analysis was performed on Drosophila L2 cells 72 hr post treatment with 1 or 10 nM of the indicated U2AF50-specific dsRNA. RNAi Kit: dsRNA prepared and purified using the MEGAscript RNAi Kit. Mega 96: dsRNA prepared with the MEGAscript Kit (Cat #1334) and purified with MEGAclear-96 (Cat. #1909).

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