- 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
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 standard single-tube MessageAmp II reaction.
Figure 3. mRNA Profiles of Mouse Tissues Stored in RNAlater. Various mouse tissues were stored in RNAlater for 1 or 4 weeks at 4°C. RNA was isolated from each tissue and analyzed using the RPA III™ Kit. 10 µg of total RNA was hybridized with 5 x 104 cpm of each of 5 combined antisense RNA probes, digested with RNase and precipitated. Products were assessed on a 5% polyacrylamide / 8 M urea gel and exposed to film for 4 hours at -80°C with an intensifying screen.
Robotic or Manual RNA Amplification using MessageAmp™II-96 aRNA Amplification Kit. Twelve replicates of HeLa cell total RNA inputs (5, 1, 0.3, and 0.1 µg) 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 3. mRNA Profiles of Mouse Tissues Stored in RNAlater
. Various mouse tissues were stored in RNAlater for 1 or 4 weeks at 4°C. RNA was isolated from each tissue and analyzed using the
Kit. 10 µg of total RNA was hybridized with 5 x 104 cpm of each of 5 combined antisense RNA probes, digested with RNase and precipitated. Products were assessed on a 5% polyacrylamide / 8 M urea gel and exposed to film for 4 hours at -80°C with an intensifying screen.
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 µg) 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.