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July 01, 2011 (Vol. 31, No. 13)

High-Throughput Gene-Expression Profiling

Automated Solutions Strive to Make Miniaturization of Genomic Applications a Reality

  • Automated qPCR Setup

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    The Access workstation easily automates the preparation of qPCR assays in 384- and 1,536-well formats for most detection platforms. Performance of the Access workstation was evaluated for preparation of 1,536-well Roche PCR microplates for qPCR analysis. 100 nanoliters of cDNA along with 500 nanoliters of probes, primers, and master mix was transferred into each well of a microplate following an automated workflow shown in the Table.

    After preparation, all plates were cycled using the Roche LightCycler® 1536 System. After analysis, the Access workstation was used to cherry-pick cDNA samples from stock plates, for all reactions with a low copy number, into 1,536-well PCR microplates to create a dilution series for further characterization. Twofold dilutions of higher concentrations of cDNA were used to identify discrete changes in mRNA expression when cycling on the LightCycler.

  • Results

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    Figure 2. Discrete twofold dilutions of cDNA show differences of approximately 1 Cp per dilution, as expected. Standard deviations for each dilution were 0.23 or less with CVs of 1.2% or less at Cp.

    Average Cp values for GAPDH probed against twofold cDNA dilutions for follow-up analysis are shown in Figure 2.

  • Summary

    The Access workstation presents exciting new capabilities for walk-away assay assembly and screening of high-throughput gene-expression analysis using real-time qPCR. Superior volumetric precision and accuracy of the Echo liquid handler ensures excellent cycle quantification with very little target DNA and lower reaction volumes.

    The flexibility of the Echo software applications to enable any-well to any-well transfer allows the user to design multifactorial assays for optimization with ease. The positional accuracy of the Echo liquid handler additionally allows for transfer with zero cross contamination. With the simplicity of the Access workstation, scientists can quickly implement an automated platform tailored for the miniaturization of gene-expression assays and other genomic applications.

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