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) .
Improved RNA Amplification and Labeling
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
Linear Results with Minimal Variablility
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
- 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.