The Ambion® MessageAmp™ Premier RNA Amplification Kit employs magnetic bead-based purification technology to remove unwanted reaction components from amplified labeled antisense RNA (aRNA). The protocol was developed using the Barnstead Lab-Line Titer Plate Shaker Model 4625, and we recommend using this shaker whenever possible. In this article we outline tips for improving recovery of MessageAmp Premier reaction products when using other (non-preferred) laboratory shakers.
High Speed and Tight Orbit Diameter Provide Optimal Magnetic Bead Agitation
The Barnstead Lab-Line Titer Plate Shaker Model 4625 is specifically recommended for the aRNA purification at the end of the MessageAmp Premier Kit
protocol. This particular shaker was chosen for its high speed and tight orbit diameter which provide a more rigorous shaking motion than other models, ensuring that samples are mixed to homogeneity for consistent, optimal sample recovery. In response to customer inquiries about using other laboratory shakers for the purification and concerns about inconsistent results, Applied Biosystems Research and Development scientists evaluated two other laboratory shakers. This article outlines tips for improving recovery of MessageAmp Premier Kit reaction products when the Barnstead Lab-Line shaker is not available.
Recommended Speed Settings
Figure 1 shows a comparison of specifications for laboratory shakers used by some MessageAmp Premier customers who have reported problems with inconsistent recovery of reaction products. The comparison illustrates that these non-preferred shakers differ considerably from the recommended Lab-Line shaker in terms of maximum speed and shaker motion diameter. It also shows the settings recommended for use in the aRNA purification protocol with the MessageAmp Premier Kit. These guidelines can serve as a basis for optimizing recovery of MessageAmp Premier Kit reaction products using other laboratory shakers with similar suboptimal speed and motion specifications.
Figure 1. Comparison of Laboratory Shaker Specifications and Recommended Speed Settings.
Recommended Protocol Modifications
The VWR DS-500 and IKA KS 260 shakers do not match the maximum rpm setting of the Barnstead Lab-Line 4625 shaker. Therefore, we recommend adding manual pipetting steps prior to agitation on the VWR and IKA shakers during the binding, washing, and elution steps of the aRNA purification to improve mixing and aRNA recovery.
For Good Recovery of aRNA, Follow the Protocol Precisely
Regardless of which shaker is used, one of the most important tips for efficient reliable aRNA recovery from the MessageAmp™ Premier aRNA purification is to add reagents in the recommended order and make sure that the sample is mixed to homogeneity with the aRNA Binding Beads at each step. For example, in steps II.F.2–3 on pages 12–13 of the manual, the protocol specifies “2. Add 60 μL aRNA Binding Mix and transfer sample to U-bottom plate.” then “3. Add 120 μL ethanol to each sample…” The transfer step not only moves samples to a container that facilitates the aRNA purification process, but also serves to mix the samples with the aRNA Binding Beads before adding the ethanol in the subsequent step. This prevents clumping of the beads to allow for maximum aRNA recovery.
Charmaine San Jose, Kelli Bramlett, Natalie S. Hernandez, Mangkey Bounpheng, Chris Willis, Quoc Hoang • Applied Biosystems