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Better RNA Amplification

NEW Amino Allyl MessageAmp II Kit now with Cy Dyes included! *

Amplify as little as 100 ng of input RNA in a single round for GeneChip analysis

Greater sensitivity due to higher yields of full-length cDNA

aRNA synthesis powered by MEGAscript, the best technology for in vitro transcription

High labeling efficiency yields consistently high percent present calls and low 3'/5' ratios in GeneChip analysis

The MessageAmp aRNA Amplification Kit was the first commercially available set of Eberwine-based aRNA amplification reagents, and the Amino Allyl MessageAmp aRNA Amplification Kit was the first kit to integrate amino allyl incorporation and aRNA amplification (see Incorporating Amino Allyl UTP During Amplification, at right). Now newer versions of both kits feature improvements that provide even greater yields of longer aRNA from inputs as small as 100 ng in a single round of amplification.

Improved RNA Amplification

The use of a modified M-MLV reverse transcriptase, ArrayScript, in conjunction with the optimization of the second-strand cDNA synthesis reaction results in increased conversion of mRNA into full-length, double-stranded cDNA templates. ArrayScript produces the highest yields of full-length cDNA compared to other reverse transcriptases (Figure 1). The second-strand cDNA synthesis reaction has been optimized for maximal conversion of first-strand cDNA into full-length double-stranded cDNA. The in vitro transcription (IVT) reaction is powered by 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 . RNA samples were amplified and labeled using both procedures and hybridized to GeneChip Human Genome Focus Arrays following the Affymetrix guidelines. Microarray expression profiling data comparing MessageAmp II to the Affymetrix recommended protocol for GeneChip analysis indicates a high level of concordance between the two procedures (Figure 2).

Figure 2. Replicate Analysis of Two Amplification Protocols. Normalized array results are plotted from replicate amplifications. Both MessageAmp II (top) and Affymetrix standard protocol (bottom) were used to amplify identical total RNA samples (1 g), and equivalent amounts of the resulting aRNA were hybridized on matched lot Human Genome Focus arrays. IVT reactions were performed for 4 hr. Correlation values are included and indicate a measure of reproducibility with both methods.

Complete Kits for Amplifying RNA and Amino Allyl Labeling

The MessageAmp II and Amino Allyl MessageAmp II aRNA Amplification Kits contain all the necessary reagents for first-strand cDNA synthesis, RNase H digestion, second-strand cDNA synthesis, cDNA purification, in vitro transcription, and aRNA purification. The Amino Allyl MessageAmp II Kit also contains reagents for amino allyl labeling, dye coupling, and labeled aRNA clean-up. Reagents for 20 amplification reactions and a detailed Instruction Manual are included with both kits.

MessageAmp II is covered by patents exclusively licensed to Incyte Genomics.

* Incorporating Amino Allyl UTP during Amplification
Dir ect incorporation of dye coupled NTPs is inefficient and results in low yields and low specific activity aRNA. This can make the cost of producing aRNA by direct incorporation very high. In addition, incorporation efficiency varies for different labels (e.g. Cy3 NTP vs Cy5 NTP). These problems can be avoided by incorporating an amino allyl NTP into the aRNA and subsequently coupling its reactive amino group with an NHS ester labeled dye (e.g. Cy or other dye). Unlike dye coupled NTPs, amino allyl modified NTPs are incorporated almost as efficiently as unmodified NTPs and are much less expensive than the dye coupled NTPs.

In the Ambion Amino Allyl MessageAmp II protocols, amino allyl UTP is incorporated during the transcription step to produce amino allyl modified aRNA. After a quick purification step, the aRNA is ready for coupling to the NHS ester label of your choice (a variety of inexpensive NHS ester dyes and other nonisotopic labels are available from Amersham Biosciences, Molecular Probes, and Pierce). After dye coupling the reaction is quenched and a final purification is performed, yielding labeled aRNA ready for hybridization.

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Ordering Information Cat# Product Name Size 1751 MessageAmp II aRNA Amplification Kit 20 rxns 1753 Amino Allyl MessageAmp II aRNA Amplification Kit 20 rxns 1795 Amino Allyl MessageAmp II with Cy3 20 rxns 1796 Amino Allyl MessageAmp II with Cy5 20 rxns 1797 Amino Allyl MessageAmp II with Cy3/Cy5 (1:3) 20 rxns 2048 ArrayScript 4000 U 2049 ArrayScript 10,000 U


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