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Matthew Cu and Zhu Zhu, Beckman Coulter, Inc.; and Roy C. Willis, Ambion, Inc. IntroductionManual target preparation for gene expression analysis is inherently laborious, time consuming and error prone. To overcome these conditions, we developed automated target preparation methods, cDNA Synthesis and in vitro Transcription, on the Biomek 3000 Laboratory Automation Workstation using the MessageAmp* II-96 aRNA Amplification Kit from Ambion*. These automated high-throughput methods are designed specifically to address problems such as variability, consistency and reproducibility associated with sample preparation. Both methods can be used to process any desired number of samples up to 96 in multiples of eight, with the start and stop on desired columns. The cDNA Synthesis method synthesizes first-strand cDNA using ArrayScript*, a reverse transcriptase (RT) engineered to produce higher yields of first-strand cDNA than wild type enzymes. ArrayScript catalyzes the synthesis of virtually full-length cDNA, ensuring superior microarray results. The cDNA undergoes second-strand synthesis, then purification to remove salts, RNA, primers and enzymes. The purified cDNA is subsequently used for in vitro transcription (IVT). To maximize the antisense RNA (aRNA) yield from the IVT reaction, Ambion uses its proprietary MEGAscript* IVT technology to generate hundreds to thousands of aRNA copies from each mRNA in a sample. The aRNA is purified using Ambion's MagMAX magnetic bead-based RNA purification to remove any unincorporated nucleotides, enzymes and salts from the aRNA prep. Quantification of aRNA is done off-line using a Molecular Devices SPECTRAmax PLUS 384 Microplate Reader. For this study, 1 µg input amount of HeLa total RNA was used for three columns each of biotin-labeled and unlabeled aRNA; all samples were prepared using 14-hour IVT reactions. Recovery of aRNA was highly consistent, with average yields of 123.1 µg and 161.2 µg for labeled and unlabeled aRNA, respectively. MaterialsBiomek 3000 and Accessories:
Consumables:
MethodsMessageAmp II-96 aRNA Amplification Protocol
Biomek 3000 cDNA Synthesis Instrument Setup
Biomek 3000 IVT Setup Instrument Setup
Biomek 3000 IVT Cleanup Instrument Setup
Results
Figure 5. RNA Amplification using MessageAmp II-96 aRNA Amplification Kit on Biomek 3000. Forty-eight replicates of 1.0 µg HeLa total RNA inputs were amplified with or without biotin according to the MessageAmp II-96 protocol on the Biomek 3000 using a 14-hour IVT reaction incubation time. Following aRNA cleanup, yield was assessed by measuring UV absorbance at 260 nm. Average yields of 123.1 µg and 161.2 µg for labeled and unlabeled aRNA, respectively. A. B. Figure 6. Bioanalyzer Electropherograms of labeled (A) and unlabeled (B) amplified cRNA from 1.0 µg input HeLa total RNA. The mRNA Smear Assay reveals the size distribution of 250 to 5500 nucleotides with a peak centered at 1400 nucleotides. Similar aRNA profiles were observed for labeled and unlabeled aRNA. Discussion and ConclusionsWe have demonstrated the automation of the MessageAmp II-96 aRNA Amplification Kit on the Biomek 3000 Laboratory Automation Workstation. This automated method produced consistent and reproducible aRNA yield with minimal user intervention. Ambion recommends an IVT reaction time of 4 – 14 hours. For the data presented here, we used a 14-hour IVT processing time for the amplification of cDNA from 1 mg input HeLa total RNA. We obtained high-yield and high-quality amplified aRNA from this automated method. The size distribution of the aRNA ranged from 250 to 5,500 nucleotides with a midpoint size of 1,400 nucleotides. We've consistently obtained these results, ensuring successful microarray analysis. * All trademarks are the property of their respective owners. For Research Use Only; not for use in diagnostic procedures.
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