Amplify as little as 100 ng of input RNA in a single round for GeneChip analysis
Includes ArrayScript, an engineered M-MLV RT, to maximize yields of full-length cDNA
aRNA synthesis powered by MEGAscript High Yield Transcription Kit, the best technology for in vitro transcription
High labeling efficiency yields consistently high percent present calls and low 3'/5' ratios in GeneChip analysis
MessageAmp II-96 aRNA Amplification Kit is the first kit designed for high throughput amplification of mRNA and is based on the patented Van Gelder-Eberwine linear RNA amplification method. This kit takes advantage of extensive improvements incorporated into the MessageAmp II aRNA Kit combined with a new magnetic bead-based purification of both the cDNA and the aRNA.
The new microplate format available with MessageAmp II-96 provides a simple, streamlined procedure for the entire aRNA amplification process, including first and second strand cDNA synthesis, in vitro transcription, and aRNA labeling. Figure 1 illustrates aRNA yields obtained with either the manual or the fully-automated MessageAmp II-96 protocol at various HeLa total RNA input levels for both 4 and 14 hour IVT reactions. Figure 2 compares the correlation coefficients (% present calls on Affymetrix Human Genome Focus Arrays) obtained using MessageAmp II-96 (Plate 1 and Plate 2) versus Ambion's st andard single-tube MessageAmp II reaction.
Figure 1. Robotic or Manual RNA Amplification using MessageAmpII-96 aRNA Amplification Kit. Twelve replicates of HeLa cell total RNA inputs (5, 1, 0.3, and 0.1 g) were processed according to the MessageAmp II-96 protocol either on the Biomek 2000 (Robot, Beckman-Coulter) or manually (human). In vitro transcription (IVT) times of (A) 14 hours and (B) 4 hours were used. Following aRNA clean-up, yield was assessed by measuring UV absorbance at 260 nm.
Figure 2. Correlation Coefficients of aRNA Data Obtained with MessageAmp II-96 Kit. HeLa cell total RNA (Control RNA included in MessageAmp II-96 aRNA Amplification Kit) was used to prepare biotin-labeled aRNA following the standard MessageAmp II-96 protocol (Plate-1 and Plate-2) and MessageAmp II protocol (tube). aRNA (10 g) was analyzed on an Affymetrix Human Genome Focus Array, and the correlation coefficients were calculated for the percent present calls.
Magnetic beads are used for both the cDNA and aRNA purification steps, eliminating the need for vacuum filtration or centrifugation. The use of beads also enables elution into smaller volumes, thus providing a higher concentration of aRNA. The entire process can be performed either manually or fully automated using a robot integrated with a thermal cycler and orbital shaker. Each kit contains sufficient reagents to amplify 100 samples, thus providing four samples for pre-testing prior to using a 96 well format.
Chris Willis, Sharmili Moturi, Xingwang Fang Ambion, Inc.