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Tutorials : Jul 1, 2011 (Vol. 31, No. 13)

High-Throughput Gene-Expression Profiling

Automated Solutions Strive to Make Miniaturization of Genomic Applications a Reality
  • Randy Dyer
  • ,
  • Celeste Glazer

Quantitative PCR (qPCR) is a powerful tool used for genotyping, SNP analysis, and gene-expression studies. Quantitative detection of small mRNA expression changes facilitates the characterization of early-stage cancer and infectious disease. Advances in qPCR technology to enable the use of high density 384- and 1,536-well microplates have led to a demand for automation and miniaturization of qPCR preparation.

With automation, researchers can generate larger sets of data quickly, while minimizing resource burden and improving reproducibility across assays. As researchers push further toward miniaturization, potential cost savings are met with a high risk for cross contamination, lower precision, and poor accuracy.

Labcyte’s Access™ workstation relies on the company’s Echo® liquid handler and its tipless, touchless transfer of samples and reagents to eliminate such risks. With the use of acoustic energy, the Echo liquid handler enables cherry-picking and reformatting of samples from any well of a source plate to any well of an assay plate with high precision, accuracy, and no risk for cross contamination.

The Access workstation seamlessly adds automated sealing, centrifugation, and bulk reagent dispensing—creating a simple walk-up and walk-away solution to overcome the challenges of miniaturized, high-throughput gene-expression profiling (Figure 1).

Cost Savings with Miniaturization

Moving qPCR workflows to high-density formats can provide a valuable increase in experimental throughput. Additionally, by reducing the amount of source mRNA consumed, researchers can dramatically increase the number of experiments performed with limited amounts of starting sample.

When commonly used volumes of 25 µL are reduced to 300 nL, the savings on master mix, probes, and primers are more than 95%. The same expenditure can yield twenty times as many high-quality assays or provide savings of tens to hundreds of thousands of dollars per year.

Simplified Protocol Development

With a suite of software applications to guide protocol development and automation control, the time required to implement the Access workstation for qPCR workflows is far less than expected. For qPCR setup, the Echo Plate Reformat (EPR) application is used to map transfers of master mix, primers, and cDNA samples from source plates to PCR assay plates. With the freedom of any-well to any-well transfers, the Echo liquid handler accommodates any arrangement of samples and assay reagents for assay design flexibility.

The Access workstation’s automation control software incorporates EPR transfer protocols into automated routines with plate-handling actions optimized for efficiency. Since every automation event is scheduled to ensure that the Echo liquid-handler steps are given the highest priority, automated routines achieve maximum throughput.

Wizard-based interfaces make it easy to edit existing protocols, track plate properties, analyze reports, and quickly recover from errors. The guidance and utility of the software alone accelerates transfer of assays from the lab bench to the Access system.

Automated qPCR Setup

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

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.