Automated Target Preparation for GeneChip* Arrays Using the Biomek® 3000-ArrayPLEX Workstation

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Zhu Zhu and Keith Roby, Beckman Coulter, Inc.

Introduction

Biomek 3000-ArrayPLEX Workstation

Microarray technology enables the simultaneous parallel analysis of changes in gene expression across the whole genome. However, the process of target preparation for the gene expression analysis is time consuming and labor intensive. We have developed the automated target preparation process using the ArrayPlex application on the Biomek 3000 Laboratory Automation Workstation with Affymetrix GeneChip Expression 3’-Amplification reagents and Agencourt® RNAClean™.

The development of the Biomek 3000-ArrayPLEX Workstation was based on the Biomek FX-ArrayPlex Workstation, a walk away automated solution based on the Biomek FX with Affymetrix GeneChip Expression 3’-Amplification reagents and QIAGEN* RNeasy* and MinElute* kits. The Biomek 3000-ArrayPLEX Workstation is comprised of a Biomek 3000 integrated with a Peltier Static Device, a Peltier Shaking Device and an optional Thermocycler. As was the case for the Biomek FX-ArrayPlex Workstation, this automated process includes three methods, cDNA Synthesis, in vitro Transcription (IVT) and Fragmentation. With fewer deck positions on the Biomek 3000, it requires some user intervention, especially when the option for an off-deck thermal cycler is chosen. However, the use of the Agencourt magnetic beads for the clean up of cDNA and cRNA simplifies the process, increases the average cRNA yields and reduces the variability from sample to sample. We believe that the Biomek 3000-ArrayPLEX Workstation offers a highly automated solution with minimal user intervention at a much lower cost.

Materials and Methods

Biomek 3000 integrated with a Biomek 3000 Peltier Shaking Device (Inheco P/N 7100018), a Biomek 3000 Peltier Static Device (Inheco P/N 7000068), an optional MJ Moto Alpha Thermocycler (MJ Research P/N RAD-0200 and PTC-200) and the DTX 880 Multimode Detector.

Reagents

Labware and Tips

Methods

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Fig. 1 Work flow of the experimental steps.

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Fig. 2 Deck layout for the cDNA synthesis step.

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Fig. 3 Deck layout for the cRNA synthesis step.

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Fig. 4 Deck layout for the cDNA and cRNA clean up steps.

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Fig. 5 Deck layout for the Fragmentation method.

The concentration of the cRNA is calculated based on the OD₂₆₀ readings measured on an offline reader. The data file of the cRNA concentration is imported back to the Fragmentation method as a guide for the normalization. The concentration of the normalized samples is examined on the offline reader for accuracy and the normalized samples are then fragmented.

Results

The Biomek 3000-ArrayPLEX Workstation allows for the processing of one full or partial 96-well plate of samples in multiples of 8 from total RNA to the biotin-labeled complementary RNA (cRNA). The decks need to be refreshed with fresh tips, labware and reagents before the cDNA clean up step in the cDNA Synthesis method and before the cRNA clean up step in the IVT method. With an off-deck thermal cycler, the reaction plate needs to be transferred to and from the thermal cycler for the incubation steps. The typical yield is 60 µg from 1 µg of HeLa total RNA and 102 µg from 2 µg of HeLa total RNA.

Table 1. The result of one experiment is shown here. A total of 24 wells of HeLa total RNA at 1 µg and 2 µg starting amount were processed. Samples were randomly distributed across the columns 1, 2 and 3 of the plate. Blue numbers represent 2 µg of HeLa total RNA and red numbers represent 2 µg of HeLa total RNA.

Conclusions

The Biomek 3000-ArrayPLEX Workstation was developed to automate the target RNA preparation process for the gene expression analysis using the Affymetrix GeneChips in the same way as the Biomek FX-ArrayPlex Workstation.

The use of the Agencourt magnetic beads in the cDNA and cRNA cleanup steps increased the average cRNA yield by nearly two times.

The option of an off-deck thermal cycler allows the end user to use their existing thermal cycler and reduce the cost of the system.

This highly automated solution requires some user intervention, but the saving of cost is significant compared to the fully automated Biomek FX-ArrayPlex Workstation.

* All trademarks are the property of their respective owners.

For Research Use Only; not for use in diagnostic procedures.

Additional Information

• ArrayPlex--Application for Microarray Target Preparation

For comments or questions about T³ Update, please contact David Daniels, Ph.D., editor.

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