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Analysis on Biomek® 3000 ArrayPlex
Target Preparation for GeneChip* Expression Analysis Using the BiomekŪ 3000 Laboratory Automation Workstation and ArrayPLEX Application
Zhu Zhu, Dan Allison, Yu Suen, Handy Yowanto, Tim Lancaster, Jeremy Smith and Keith Roby,
Beckman Coulter, Inc., Fullerton, California.
Microarrays have revolutionized gene expression analysis. It is critical to obtain quality target RNA with high level of reproducibility to enable the standardization of the GeneChip assays that usually involves multiple operators and laboratories. We have developed a solution that reduces the variability from human error and saves time and labor using the ArrayPLEX application on the Biomek 3000 (B3K) Laboratory Automation Workstation.
The ArrayPLEX application is part of the GeneomeLab family of applications (Beckman Coulter).
It is derived from the ArrayPlex application on the Biomek FX workstation (BFX), a walk-away automated solution for microarray target preparation. The B3K system with the ArrayPlex application is comprised of a B3K, a Peltier Static Device and a Peltier Shaking Device. The application allows for the deck of the B3K to be configured for a customer-provided thermocycler. The highly automated process includes three methods: cDNA Synthesis, IVT and Fragmentation. A full or partial 96-well plate of samples can be processed in multiples of eight in a single run. Affymetrix*’ GeneChip Expression 3’-Amplification reagents were used for cDNA synthesis and cRNA amplification and labeling. The magnetic bead-based Agencourt RNAClean kit, that provides a simple and highly reproducible process for efficiently recovering quality nucleic acids, was used during cDNA and cRNA cleanup. In this study, 10 plates of 24 samples with 1 µg or 2 µg of human HeLa total RNA were processed using the automated methods. Forty-eight of the amplified cRNA samples were randomly selected to hybridize to the Affymetrix Human Genome U133 Plus 2.0 Arrays*.
Features:
• The ArrayPLEX application was performed on the B3K, a small cost-effective platform, to automate the target RNA preparation process for gene expression analysis using the Affymetrix GeneChip.
• Agencourt® RNAClean™ for the cDNA and cRNA cleanup steps, replace filtration-based methods
• Target RNA preparation from total RNA samples in full 96-well plates is20 hours and partial 96-well plates with multiples of 8 is 15.5 hours.
• A high and consistent yield was obtained using the magnetic bead-based Agencourt RNAClean kit. The average yields were 83.1 µg for the 1µg total RNA and 135.6 µg for the 2 µg total RNA starting materials.
• Bioanalyzer electropherograms showed similar non-degraded profiles for cRNA generated from 1 µg and 2 µg of total RNA, indicating no performance bias with different sample concentrations.
• Forty-eight cRNA samples were hybridized to the Affymetrix Human Genome U133 Plus 2.0 Array. The data show good correlations and reproducibility between samples that were prepared on different workstations.
• The overall validation data pass the specifications set by Affymetrix.
* All trademarks are the property of their respective owners. Where applicable, PCR is covered by patents owned by Hoffman-LaRoche Inc.
