- Increased amplification – Up to 5,000-fold
- Greater sensitivity due to increased yields of full-length cDNA
- GeneChip® quantities of aRNA from only 100 ng input RNA
- Efficient labeling produces high percent present calls and low 3'/5' ratios
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MessageAmp™ II vs. Affymetrix Protocol
We assessed the reproducibility and consistency of the MessageAmp II protocol compared to the Affymetrix recommended protocol. cRNA samples were amplified and labeled using both procedures and hybridized to GeneChip® Human Genome Focus Arrays following the Affymetrix guidelines. To view the results of these experiments please click here. |
Expression profiling using gene arrays to simultaneously measure the expression levels of thousands of genes in a single experiment has become the most widely used method for assessing gene expression on a genome wide scale. The primary limitation of this technology is the requirement for large amounts of RNA for array hybridization. This is especially a problem when working with limited samples, such as small biopsies, mRNA-deficient cells and tissues, primary cell culture, and laser capture microdissection (LCM) samples. This hurdle is usually overcome by using T7 RNA Polymerase driven transcription of cDNA obtained from total RNA samples to amplify sufficient amounts of cRNA. This amplification method is commonly referred to as the “Eberwine method” (1), “IVT method” or the “aRNA or cRNA method”.
Ambion’s MessageAmp™ aRNA Amplification Kit was the first commercially available set of Eberwine-based aRNA amplification reagents. We are now pleased to announce the release the of the new MessageAmp™ II aRNA Amplification Kit that incorporates extensive improvements to the original kit. (For comparison to the original kit, see article sidebar entitled MessageAmp II Concordance with Original MessageAmp Kit.) At Ambion, we recognize that improving cRNA amplification involves more than simply increasing yields of labeled cRNA. As evident from the microarray expression profiling data we have generated comparing MessageAmp II to the Affymetrix recommended protocol for GeneChip® analysis, a high level of concordance is seen between the two procedures. In addition, the new kit provides a wide dynamic range of RNA input (100 pg – 5 µg). Furthermore, we have found that a single round of amplification performed with MessageAmp II on samples as low as 100 ng generates sufficient cRNA for GeneChip analysis.
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Factors that can Affect cRNA Quality and Yield
While developing MessageAmp™ II we performed a number of experiments investigating various criteria that can impact the quality and yields of cRNA synthesized with the MessageAmp II kit. These include the effect of RNA quality, total RNA input, IVT incubation times and the number of rounds of amplification. To view the results of these experiments please click here. |
MessageAmp II: What’s New and Improved?
While developing MessageAmp II we realized early on that the reverse transcription step and the second strand cDNA synthesis reaction are critical for generating high yields of labeled cRNA with minimal change to the initial representation of message abundance in total RNA samples. This led to the development of ArrayScript™, a specifically engineered M-MLV reverse transcriptase and optimization of the second strand cDNA synthesis reaction.
ArrayScript™: Engineered M-MLV RT for Improved Yields of Full-length cDNA
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 enzymes (Figure 3). In fact, experiments performed at Ambion have also demonstrated that up to twice as much cRNA is synthesized when the first-strand cDNA is generated with ArrayScript compared to wild-type M-MLV or AMV, and other engineered M-MLV reverse transcriptases (Figure 4) when low amounts of total RNA are used (~ 100 ng). This can eliminate the need to perform two rounds of cRNA amplification when working with low amounts of total RNA, thus reducing the cost and time of the reaction when using MessageAmp II.
Second-strand cDNA Synthesis
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.
An Improved Kit for cRNA Amplification
The use of 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. In addition, since the in vitro transcription (IVT) reaction is powered by MEGAscript® technology, the IVT incubation times are shortened, thus enabling more reliable access to expression information from total RNA samples of less than 1 µg. These improvements in the MessageAmp II Kit also carry through to two-round reactions, facilitating robust amplification of total RNA from samples as low as 100 pg.
The MessageAmp II aRNA Amplification 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 included.
MessageAmp™ II-96 aRNA Amplification kit
The MessageAmp II-96 aRNA Amplification Kit is the first kit designed for high throughput amplification of messenger RNA. It is based on the patented Eberwine linear RNA amplification method. This new kit takes advantage of the extensive improvements incorporated into the MessageAmp II kit combined with a new magnetic bead-based purification of both the cDNA and the aRNA. The magnetic bead-based purification reduces elution volumes for easier protocol integration.
These improvements offer:
- Consistently higher yields with magnetic bead compared to filter based method
- Highly reproducible aRNA yields and size (CV less than 10%)
- Flexible format allows RNA amplification with a partial to a full 96 well plate
- Easily automated for high-throughput amplification of mRNA using robotics
Downloadable protocols for the use of this kit with specific liquid handling systems are available at our Automation Resource.
The kit supplies reagents for single round amplification only. Each kit contains sufficient reagents to amplify 100 samples, allowing up to 4 samples to be pre-tested before moving to the 96 well format.
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.
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.
References
1. 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.
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