- Maximum Flexibility — 20 single round or 10 two round amplifications
- cRNA synthesis, powered by MEGAscript®, ensures best representation of gene expression profile
- Greater sensitivity due to higher yields of full length cDNA
- Up to 4,000 fold amplification
The Amino Allyl MessageAmp™ aRNA Amplification Kit was the first kit that integrated amino allyl incorporation and aRNA amplification. The new Amino Allyl MessageAmp II aRNA Amplification Kit incorporates extensive improvements to the original kit, as well as the inclusion of the Cy™ dyes. The Cy dyes are supplied in individual tubes sufficient for one labeling reaction.
Improved First- and Second-strand cDNA Synthesis
Scientists at Ambion have rationally engineered the wild type M-MLV reverse transcriptase such that the modified enzyme, ArrayScript™, produces as high or higher yields of full-length cDNA compared to other reverse transcriptases. The second-strand cDNA synthesis reaction has also been optimized to be compatible with the first-strand cDNA products generated with ArrayScript to allow maximal conversion of first-strand cDNA into full-length double-stranded cDNA templates.
Increased Labeling Efficiency of Your aRNA
Several different methods can be used to label RNA for array analysis. Labels 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 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. Cy™3 NTP vs Cy™5 NTP). 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. 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.
Incorporating Amino Allyl UTP 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. RNase H included during the second-strand synthesis reaction cleaves the mRNA template into small fragments, which serve as primers during this step, producing double-stranded cDNA. The cDNA is purified away from contaminating rRNA, mRNA and primers and is then used in a large scale in vitro transcription reaction powered by Ambion’s patented MEGAscript® technology. 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 (Cy label included in some kit formats; see ordering information above). The coupling reaction is quenched, a final purification is performed, and the labeled aRNA is ready for hybridization.
A Complete Kit for Amplifying Amino Allyl Labeled aRNA
The Amino Allyl MessageAmp II aRNA Amplification Kit comes with all reagents for reverse transcription, second-strand cDNA synthesis, RNase H digestion, cDNA purification, in vitro transcription, aRNA purification, amino allyl labeling, dye coupling, and labeled aRNA clean-up. Cy dye is included in some kit formats. Reagents for 20 amplification reactions and a detailed Instruction Manual are included.
Amino Allyl MessageAmp™ II-96
The new Amino Allyl MessageAmp™ II-96 aRNA Amplification Kit is the first kit for high throughput amplification of messenger RNA that incorporates an amino allyl UTP into the newly synthesized RNA to facilitate labeling of the RNA. The kit combines the extensive improvements incorporated into our new Amino Allyl MessageAmp™ II Kit with a magnetic bead-based purification of both the cDNA and aRNA. This method of purification reduces elution volumes for easier protocol integration.
Features of the kit include:
- Consistently higher yields with magnetic bead- compared to filter-based purification
- Highly reproducible aRNA yields and size (CV less than 10%)
- Flexible format allowing RNA amplification with a partial or full 96 well plate
- Simple integration into robotic platforms
The kit contains sufficient reagents for single round amplification of 100 samples (allowing up to 4 samples to be pre-tested before moving to the 96 well format).
96-Well Magnetic-Ring Stand
Ambion’s 96-Well Magnetic-Ring Stand features 96 powerful ring-shaped magnets arranged to cradle each well of a 96-well plate to capture beads thoroughly and quickly. Captured magnetic beads will form a donut-shaped pellet with a large hole in the center. This capture pattern facilitates both supernatant removal and subsequent bead resuspension. The stand is suitable for high throughput applications conducted manually with multichannel pipettors or with robotic liquid handlers.
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