- NOTE: This kit will be discontinued on January 1, 2010. Please use MessageAmp Premier Kit in place of this kit.
- Representational amplification of small amounts of RNA for array analysis
- Based on the patented Eberwine aRNA amplification procedure
- Amplify mRNA 1000X in a single round of amplification
- Incorporates MEGAscript™ high yield transcription technology
NEW! See MessageAmp™ Premier: An Improved Kit for aRNA Amplification
The new MessageAmp Premier aRNA Amplification Kit is the next generation of Ambion biotin labeling/amplification kits to support microarray technology, such as the Affymetrix GeneChip® platform. It incorporate extensive improvements, including a simplified workflow and more efficient reactions. Perform expression profiling with as little as 20 ng of input RNA or 100 ng of total RNA.
The MessageAmp procedure is based on antisense RNA (aRNA) amplification first described by Van Gelder and Eberwine (1), and involves a series of enzymatic reactions resulting in linear amplification of exceedingly small amounts of RNA for use in array analysis. Unlike exponential RNA amplification methods, such as NASBA and RT-PCR, aRNA amplification maintains representation of the starting mRNA population (2).
The procedure begins with total or poly(A) RNA that is reverse transcribed using a primer containing both oligo(dT) and a T7 RNA polymerase promoter sequence (see schematic at left). After first-strand synthesis, the reaction is treated with RNase H to cleave the mRNA into small fragments. These small RNA fragments serve as primers during a second-strand synthesis reaction that produces a double-stranded cDNA template for transcription. Contaminating rRNA, mRNA fragments and primers are removed and the cDNA template is then used in a large scale in vitro transcription reaction to produce linearly amplified aRNA. The aRNA can be labeled with biotin rNTPS or amino allyl-UTP during transcription. Alternatively, unlabeled aRNA can be used as a template for a reverse transcription with CyDye™-labeled dNTPs to generate labeled cDNA. The RETROscript™ Kit is ideal for this purpose. For increased yields, the aRNA can also be used as template for cDNA synthesis followed by a second round of amplification using MessageAmp.
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How is 10 µg aRNA from 1 µg Total RNA 1000X Amplification?
If mRNA comprises 1% of total RNA, 1 µg total RNA contains 10 ng mRNA. A single MessageAmp reaction, starting with 1 µg total RNA, will yield a minimum of 10 µg aRNA. 10 µg is 1000X more than 10 ng. |
The MessageAmp Kit contains reagents optimized for each step of the aRNA procedure and includes the patented MEGAscript™ technology for high-yield in vitro transcription. While yields of aRNA will vary depending on RNA source and quality, a typical reaction will yield as much as 10 µg of aRNA starting from 1 µg of total RNA (this represents a 1000X amplification). Smaller amounts of total RNA (10 ng) can be used with two cycles of amplification to generate 10 µg or more of aRNA.
The MessageAmp aRNA Kit contains all the necessary reagents for first-strand cDNA synthesis, RNase H digestion, second-strand synthesis, cDNA purification, in vitro transcription and aRNA purification. Reagents for 20 reactions and a detailed Instruction Manual are also included.
Bio-11-UTP and Bio-16-UTP
Ambion’s biotinylated UTPs are ideal for use as substrates in in vitro transcription reactions. These modified nucleotides can be utilized by a variety of RNA polymerases, including T7, T3, and SP6 RNA polymerases, as well as other RNA modifying enzymes. Biotinylated RNA can be used in place of radioactively labeled RNA in many applications, including cRNA amplification and labeling for microarray target preparation. Detection is then carried out via one of a variety of streptavidin-based methods. Ambion's Bio-UTPs are confirmed free of non-specific endonuclease activity, exonuclease activity, and RNase activity and are functionally tested using the MessageAmp™ II aRNA Amplification Kit.
References
- Van Gelder, R.N., von Zastrow, M.E., Yool, A., Dement, W.C., Barchas, J.D. and Eberwine, J.H. (1990) Amplified RNA synthesized from limited quantities of heterogeneous cDNA. Proc Natl Acad Sci USA 87: 1663-1667.
- Poirier, G.M., and Erlander, M.G. (1998) Postdifferential Display: Parallel processing of candidates using small amounts of RNA. METHODS: A Companion to Methods in Enzymology 16: 444-452.
Trademarks, Patents, Licensing
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