aRNA Synthesis and Labeling for Array Analysis
Amino Allyl MessageAmp™ II aRNA Amplification Kits--
Maximum flexibility
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Labeling aRNA
Several different methods can be used to label amplified antisense RNA (aRNA) for array analysis. Cy dyes, other fluorophores, or biotin can be incorporated: 1) during the in vitro transcription (IVT) step of aRNA synthesis via modified-rNTP incorporation (direct labeling), 2) during a subsequent reverse transcription reaction via modified-dNTP incorporation (direct labeling), or 3) in a coupling reaction after aRNA synthesis where the label is added to an amino allyl-modified rNTP incorporated during IVT or reverse transcription (indirect labeling). Increasingly, amino allyl incorporation combined with a secondary dye coupling reaction is being used for the generation of samples for array analysis.
Advantages of Indirect Labeling
Direct incorporation of labeled 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 indirect labeling, whereby an amino allyl NTP is incorporated into the aRNA, and the reactive amino group is subsequently coupled with an NHS ester label (e.g. biotin, Cy dye). Compared to dye-coupled NTPs, amino allyl modified NTPs are incorporated almost as efficiently as unmodified NTPs by ArrayScript Reverse Transcriptase (Cat# 2048) and are much less expensive. Figures 1 and 2 demonstrate equivalent labeling with Cy3 and Cy5 dyes using the Amino Allyl MessageAmp™ II aRNA Amplification Kit.
Figure 1. aRNA Yields From Amino Allyl MessageAmp™ II aRNA Amplification Kit. The indicated amounts of Control RNA (HeLa cell total RNA) were amplified for the in vitro transcription (IVT) incubation times indicated. While longer IVT incubation times produced more aRNA, the amount of aRNA required for most microarrays was obtained with a four hour incubation with ≥500 ng of input RNA. This data was obtained using the Control RNA provided with the Amino Allyl MessageAmp II aRNA Amplification Kit. Note that aRNA yields can vary considerably with sample quality.
Figure 2. Scatter Plot Demonstrating Equivalent Labeling. Scatter plot showing signal intensities of Cy™3 vs. Cy™5 labeled aRNA synthesized with the Amino Allyl MessageAmp™ II aRNA Kit.
Figure 1. aRNA Yields From Amino Allyl MessageAmp™ II aRNA Amplification Kit. The indicated amounts of Control RNA (HeLa cell total RNA) were amplified for the in vitro transcription (IVT) incubation times indicated. While longer IVT incubation times produced more aRNA, the amount of aRNA required for most microarrays was obtained with a four hour incubation with ≥500 ng of input RNA. This data was obtained using the Control RNA provided with the Amino Allyl MessageAmp II aRNA Amplification Kit. Note that aRNA yields can vary considerably with sample quality.
Figure 2. Scatter Plot Demonstrating Equivalent Labeling. Scatter plot showing signal intensities of Cy™3 vs. Cy™5 labeled aRNA synthesized with the Amino Allyl MessageAmp™ II aRNA Kit.
Now Available with Cy dyes
For versatility and convenience, the new Amino Allyl MessageAmp II aRNA Kit can be ordered with Cy3, Cy5, Cy3 and Cy5, or without label. Other nonisotopic labels, available from Amersham Biosciences, Molecular Probes, and Pierce, can also be coupled to the amino allyl moiety.
Improved Kit for High Yield and High Reproducibility
The Amino Allyl MessageAmp aRNA Amplification Kit was the first kit that integrated amino allyl incorporation with RNA amplification. The new Amino Allyl MessageAmp II aRNA Kit includes extensive improvements to the original kit. Both kits contain all the components needed to perform RNA amplification, including reagents for first- and second-strand cDNA synthesis, cDNA purification, large scale in vitro transcription, and aRNA purification. Additionally, both kits include Ambion's highly purified amino allyl UTP, and reagents for Cy dye coupling and clean-up. The final purification step has been optimized for thorough removal of excess dye to ensure very low background signal on arrays.
The Amino Allyl MessageAmp II aRNA Amplification Kit uses ArrayScript to maximize conversion of first-strand cDNA into full-length double-stranded cDNA templates. Buffers have been modified to increase labeling efficiency, and each step of the protocol has been optimized for high yields and reproducibility. Starting with 2 µg of total RNA (~60 ng mRNA), a single round of amplification using the Amino Allyl MessageAmp Kit will yield up to 150 µg aRNA. This represents a 2500-fold amplification of mRNA. In addition, as little as 10 ng total RNA (300 pg mRNA) can be amplified in a single round to produce more than 1 µg aRNA.
Amino Allyl MessageAmp is covered by patents exclusively licensed to Incyte Genomics
The Amino Allyl MessageAmp II aRNA Amplification Kit uses ArrayScript to maximize conversion of first-strand cDNA into full-length double-stranded cDNA templates. Buffers have been modified to increase labeling efficiency, and each step of the protocol has been optimized for high yields and reproducibility. Starting with 2 µg of total RNA (~60 ng mRNA), a single round of amplification using the Amino Allyl MessageAmp Kit will yield up to 150 µg aRNA. This represents a 2500-fold amplification of mRNA. In addition, as little as 10 ng total RNA (300 pg mRNA) can be amplified in a single round to produce more than 1 µg aRNA.
Amino Allyl MessageAmp is covered by patents exclusively licensed to Incyte Genomics
