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Better RNA Amplification

href="http://www.ambion.com/catalog/CatNum.php?1334">MEGAscript technology, requiring shorter IVT incubation times, thus enabling more reliable access to expression information from total RNA samples of less than 1 g. Both kits are effective with a wide dynamic range of RNA input (100 pg-5 g). A single round of amplification performed with MessageAmp II on samples as small as 100 ng results in up to 5000 fold amplification, generating sufficient aRNA for GeneChip analysis. These improvements in the MessageAmp II Kit also carry through to two-round reactions, facilitating robust amplification of total RNA from samples as small as 100 pg.

Figure 1. ArrayScript Reverse Transcriptase in MessageAmp II Produces Up to Twice the aRNA Yield. A comparison of ArrayScript RT (AS; 200 U), Competitor RT (Comp; 200 U); wild type M-MLV RT (200 U), and wild type AMV RT (10 U) using MessageAmp II conditions demonstrates that ArrayScript outperforms other commercial RTs. 90 ng HeLa-S3 total RNA (28S/18S rRNA ratio = 1.7) was heat denatured in the presence of T7 oligo(dT) at 70C for 10 min. Reverse transcription was initiated with the RT enzyme and incubated at 42C for 2 hr. Following first strand cDNA synthesis, DNA polymerase was added for second strand synthesis, which was incubated at 16C for 2 hr. The double-stranded cDNA was then filter purified and added to a 4 hr in vitro transcription reaction. The resulting aRNA was purified and analyzed by NanoDrop Spectrophotometer.


MessageAmp II vs. Affymetrix Protocol

We assessed the reproducibility and consistency of the MessageAmp II protocol compared to the protocol recommended by Affymetrix
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