Automated Workflow: RNA Amplification and Labeling for Microarray Analysis
| Ambion MessageAmp™ Premier RNA Amplification Kit (Applied Biosystems) Sciclone ALH 3000 Workstation (Caliper Life Sciences) Applied Biosystems Custom Solutions The Ambion MessageAmp™ Premier RNA Amplification Kit is the latest innovation for preparation of RNA samples for microarray analysis using Affymetrix® GeneChip® Arrays. This article describes how the kit protocol can be completely automated using the Caliper Sciclone ALH 3000 Workstation to produce high quality aRNA from 8 to 96 samples in a single day (overnight for low RNA inputs) [1]. |
${comergentUrl}
Improved RNA Amplification and Labeling
The MessageAmp Premier RNA Amplification Kit is based on the linear RNA amplification method pioneered in the Eberwine laboratory [2], with major enhancements that create a simplified workflow, improved performance (e.g., up to 50,000-fold amplification), and flexible RNA input requirements (e.g., 20–500 ng total RNA). Compared to glass fiber filter-based purification methods, the magnetic bead-based aRNA purification used in the MessageAmp Premier Kit increases aRNA yields ~25% and makes the procedure more amenable to automation for high-throughput applications. Figure 1 compares the MessageAmp Premier Kit protocol with current RNA amplification protocols to highlight the workflow improvements.
Figure 1. The Streamlined Ambion MessageAmp™ Premier Kit Employs Optimized Reagent Formulations for Improved Workflow. Initial primer annealing and cDNA purifications steps have been eliminated. The second-strand cDNA synthesis reaction has been reduced to 1 hr. The final aRNA purification step using magnetic beads is more efficient and requires less sample handling. The total time saved with the MessageAmp Premier Kit is up to 3.5 hr per sample, depending on the amount of input RNA and IVT reaction time.
Figure 1. The Streamlined Ambion MessageAmp™ Premier Kit Employs Optimized Reagent Formulations for Improved Workflow. Initial primer annealing and cDNA purifications steps have been eliminated. The second-strand cDNA synthesis reaction has been reduced to 1 hr. The final aRNA purification step using magnetic beads is more efficient and requires less sample handling. The total time saved with the MessageAmp Premier Kit is up to 3.5 hr per sample, depending on the amount of input RNA and IVT reaction time.
Automating the Workflow
The Caliper Life Sciences Sciclone ALH 3000 Workstation was programmed to perform the RNA amplification and labeling procedure as indicated in the MessageAmp Premier Instruction Manual. The in vitro transcription reaction was incubated for 14 hr and aRNA was eluted in 50 µL at the end of the procedure. The workstation employed the Z-8 robotic arm to distribute reagents, a high-volume head for cleanup, a side-mounted Twister II robotic arm to move sample plates, and a thermal cycler for all reaction incubations. Temperature-sensitive reagents were stored on a Peltier cooling block on the deck of the workstation.
Linear Results with Minimal Variablility
Increasing RNA input amounts (20 ng, 50 ng, and 100 ng) were amplified and labeled with 16 replicates to evaluate the consistency and linearity of aRNA yields using the MessageAmp Premier Kit on the Sciclone ALH 3000 Workstation (Figure 2). aRNA yield from the replicate reactions exhibited minimal variability and increased linearly as the amount of input RNA increased. With a 14-hour IVT reaction, as little as 20 ng of input RNA produced sufficient aRNA for a microarray gene expression study. The median aRNA size distribution, assessed on an Agilent 2100 bioanalyzer, was consistent among the replicates and input RNA amounts (Figure 3).
Figure 2. Automation of the Ambion MessageAmp™ Premier RNA Amplification Kit Produces Sufficient aRNA for Gene Expression Studies. An automated program developed for the Caliper Life Sciences Sciclone ALH 3000 Workstation enabled the MessageAmp Premier Kit to amplify HeLa Total RNA (16 replicates each for 20, 50, and 100 ng input amounts) in an overnight experiment. The in vitro transcription time was 14 hr, and yield was assessed by measuring absorbance at 260 nm (Panel A). There was a linear correlation between yield and input amount of RNA (Panel B).
Figure 3. Automation of the Ambion MessageAmp™ Premier RNA Amplification Kit Produces High Quality aRNA for Gene Expression Studies. An automated program developed for the Caliper Life Sciences Sciclone ALH 3000 Workstation enabled the MessageAmp Premier Kit to amplify HeLa Total RNA (16 replicates each for 20, 50, and 100 ng input amounts) in an overnight experiment that included a 14 hr in vitro transcription reaction. Panel A. The Median size of aRNA resulting from the indicated input amounts. Panel B. Resulting aRNA size was assessed with an Agilent 2100 bioanalyzer. Ladder: RNA 6000 Ladder; marker lengths are 0.2, 0.5, 1.0, 2.0, 4.0, and 6.0 kb.
Figure 2. Automation of the Ambion MessageAmp™ Premier RNA Amplification Kit Produces Sufficient aRNA for Gene Expression Studies. An automated program developed for the Caliper Life Sciences Sciclone ALH 3000 Workstation enabled the MessageAmp Premier Kit to amplify HeLa Total RNA (16 replicates each for 20, 50, and 100 ng input amounts) in an overnight experiment. The in vitro transcription time was 14 hr, and yield was assessed by measuring absorbance at 260 nm (Panel A). There was a linear correlation between yield and input amount of RNA (Panel B).
Figure 3. Automation of the Ambion MessageAmp™ Premier RNA Amplification Kit Produces High Quality aRNA for Gene Expression Studies. An automated program developed for the Caliper Life Sciences Sciclone ALH 3000 Workstation enabled the MessageAmp Premier Kit to amplify HeLa Total RNA (16 replicates each for 20, 50, and 100 ng input amounts) in an overnight experiment that included a 14 hr in vitro transcription reaction. Panel A. The Median size of aRNA resulting from the indicated input amounts. Panel B. Resulting aRNA size was assessed with an Agilent 2100 bioanalyzer. Ladder: RNA 6000 Ladder; marker lengths are 0.2, 0.5, 1.0, 2.0, 4.0, and 6.0 kb.
Reduced Handling, Increased Speed
Automated aRNA synthesis reactions using the MessageAmp Premier Kit produced aRNA of sufficient yield and quality for use in subsequent gene expression studies. With its reduced number of steps, single-tube format, and unsurpassed performance, the MessageAmp Premier Kit reduces total reaction time and reagent consumption while improving yield of labeled RNA. Automating this method on the Sciclone ALH 3000 Workstation greatly decreases labor time—the reactions are simply set up and the instrument does the rest of the work, producing purified, labeled aRNA ready for use.
References
- This work was presented at the Lab Automation meeting in Palm Springs, CA, Jan 27–30, 2008.
- Van Gelder RN, von Xastrow ME, Yool A, Dement DC, Barchas JD, Eberwine JH (1990) Amplified RNA synthesized from limited quantities of heterogeneous cDNA. Proc Natl Acad Sci USA 87:1663–1667.
