| Synthesize Labeled aRNA
for Array Analysis
Amino Allyl MessageAmp™ aRNA Kit Ambion's Amino Allyl MessageAmp™ aRNA Kit includes all the reagents needed for RNA amplification using the Van Gelder and Eberwine method1 along with components for the incorporation of amino allyl UTP for secondary labeling of the amplified RNA with a succinimide (NHS) ester dyes. �• Efficient incorporation of amino allyl UTP into aRNA for coupling with Cy™, Alexa Fluor®, and other NHS ester dyes and nonisotopic labels • More efficient and less expensive than direct incorporation of labeled NTPs during aRNA synthesis • Amplify mRNA 2000X or more for array analysis Increased Labeling Efficiency
of Your aRNA
Several different methods can be used to label RNA for array analysis. Cy dyes, other fluorophores, or biotin can be incorporated: 1) during the in vitro transcription step of aRNA synthesis, 2) during a subsequent reverse transcription reaction or, 3) after aRNA synthesis in a post-synthesis coupling reaction. Increasingly, amino allyl incorporation combined with a secondary dye coupling reaction is used for the generation of probes for array analysis. This strategy offers several advantages over the direct incorporation of labeled NTPs. Direct incorporation of labeled NTPs is inefficient, resulting in low yields and low specific activity aRNA. In addition, incorporation efficiency varies for different labels (e.g. Cy3 NTP vs Cy5 NTP). Because many labeled NTPs are incorporated inefficiently, the cost of producing labeled aRNA can be very high. These problems can be avoided by incorporating an amino allyl NTP into the aRNA and subsequently coupling its reactive amino group to an NHS ester label (e.g. biotin, Cy 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. A variety of inexpensive NHS ester dyes and other nonisotopic labels, are available from Amersham Biosciences, Molecular Probes and Pierce. Optimized Kit for High Yield and High Reproducibility
The Amino Allyl MessageAmp aRNA Kit is the first kit that integrates amino allyl incorporation and aRNA amplification. The kit contains all components needed to perform aRNA amplification, including reagents for first- and second-strand cDNA synthesis, cDNA purification, large scale in vitro transcription and aRNA purification. Additionally, the kit includes Ambion's highly purified amino allyl UTP and reagents for dye coupling and clean up. Each step of the protocol has been optimized for high yields and reproducibility. The final purification step has been optimized for thorough removal of excess dye to ensure very low background signal on your arrays. Starting with 2 µg of total RNA (containing ~60 ng mRNA) a single round of amplification using the Amino Allyl MessageAmp Kit will yield up to 150 µg of aRNA. This represents a 2500X amplification of mRNA. As little as 10 ng total RNA (300 pg mRNA) used as template in a single round of amplification typically produces more than a microgram of aRNA. Incorporating Amino Allyl During Amplification
The aRNA amplification procedure begins with total or poly(A) RNA that is reverse transcribed using an oligo(dT) primer containing a T7 RNA polymerase promoter sequence, as illustrated in Figure 1. | | Figure 1. Amino Allyl MessageAmp™ Procedure. | After first-strand cDNA synthesis, the reaction is treated with RNase H to cleave the mRNA template into small fragments. The resulting small RNA fragments serve as primers during second-strand cDNA synthesis, producing double-stranded cDNA that will serve as template for the subsequent in vitro transcription reaction. Contaminating rRNA, mRNA fragments and primers are removed and the purified cDNA is then used in a large scale in vitro transcription reaction. This transcription reaction uses Ambion's patented MEGAscript™ large scale in vitro transcription technology to maximize yield. 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. The coupling reaction is quenched, a final purification is performed, and the labeled aRNA is ready for hybridization. The amplification and labeling protocol can be completed in less than one day. In addition to the Amino Allyl MessageAmp aRNA Kit, Ambion also offers the MessageAmp aRNA Kit that includes all necessary reagents for aRNA amplification (without reagents for amino allyl labeling). Also available is the Amino Allyl cDNA Labeling Kit for labeling cDNA for use when the starting RNA sample does not require amplification. |