featuring the Echo Acoustic Technology
TITLES and AUTHORS
To facilitate large-scale, high-throughput functional genomics studies using RNAi, we have developed acoustic droplet ejection (ADE) technology to meet the current demands of a successful siRNA library-based screen. High throughput applications require reliable and reproducible transfections to be performed in high-density well plate formats. The success of an siRNA experiment relies on the effective delivery of the siRNA molecule. Here we have compared the performance of the ADE technology for standard transfection, reverse transfection and Accell (Thermo Scientific) siRNA molecules against more traditional liquid handling methods.
Quantitative PCR (qPCR) has enabled a wide range of real-time applications including comparisons in gene expression, genotyping and SNP analysis. Reductions in scale reduce sample and reagent volumes and therefore total running and operational costs. To ensure high data quality, the liquid handling employed in low volume reactions must be exceptionally precise and accurate. This application note highlights the capabilities of the Echo liquid handler to dispense into 384-well qPCR plates with total reaction volumes as low as 250 nanoliters. Standard deviations of less than 0.25 and CVs of less than 2% are seen routinely across plates using the low volume dispensing of the Echo liquid handler. The ability of the Echo liquid handler to transfer from any well to any well simplifies assay setup. The results demonstrate the capabilities to enable assay setup with flexible plate layouts, reduce reagent volumes and increase throughput.
Aqueous enzyme solutions have historically been problematic to dispense in tip-based liquid handlers. Enzymes are often supplied in solution containing glycerol. Glycerol increases the viscosity of the solution, and decreases tip-based pipette performance as the sample tends to stick to both inside and outside of the tip. This reduces accuracy and increases imprecision in lownanoliter volume transfers. Furthermore, aqueous enzyme solutions cause the meniscus within the well to lay highly tilted in some wells especially after centrifugation of well plates. The Echo® 555 liquid handler (Labcyte Inc.) utilizing acoustic droplet ejection (ADE) technology was used to accurately and precisely dispense highly viscous aqueous enzyme solutions. With the ability to recognize and adjust for tilted meniscus, the Echo 555 liquid handler was able to transfer 50 to 200 nanoliter volumes with assay CVs ranging from 3.2% to 4.6%. Use of ADE reduced valuable enzyme waste through elimination of tips while improving accuracy and precision. These experiments also open possibilities for total assay assembly using tipless, touchless high-throughput acoustic droplet ejection technology.
Large-scale, high-throughput siRNA experiments require a transfer system that can facilitate reliable and reproducible transfections in high-density well-plate formats, as well as effective delivery of the siRNA molecule. This study demonstrates superior performance of the Echo liquid handler compared to more traditional liquid handling methods for standard, reverse and lipid-free transfection of siRNA molecules into mammalian cells.Results of the study clearly demonstrate the ability of the Echo liquid handler to deliver comparable transfection efficiency, knock-down and cell morphology to reactions prepared using traditional pipeting methods. In fact, reactions prepared with the Echo liquid handler were miniaturized to use significantly less volume of siRNA and other reagents, and still yielded more representative inclusion intensities than traditional manual pipetting techniques. Such miniaturization can result in significant cost savings and greater confidence in the results from siRNA screening studies.
Acoustic droplet ejection (ADE) is a completely touchless transfer technology for liquid handling that eliminates the use of disposable tips and wash cycles. The ability to transfer from any well of the source plate to any well of the destination plate enables flexible assay setup in both 384-well and 1536-well plates. For the ADE, we used the Labcyte Echo® 555 liquid handler (Labcyte Inc.) to transfer different volumes of aqueous-based reagents from several commonly used biochemical assay kits. To measure accuracy and precision, each reagent was doped with 0.15 nM fluorescein so that the transferred volumes could be measured on an Envision reader (PerkinElmer). The volumetric precision for each reagent transferred was below 2.5% CV and the accuracy was within 10% of the expected volume. These results demonstrate that ADE provides precision and accuracy for a newly expanded range of fluid types that is comparable to established performance with DMSO. These new capabilities enable assay setup with flexible plate layout and volume reduction for reagent and consumables savings and higher throughput.
Disposable pipette tips can cause errors in dose-response experiments. More accurate dose-response curves can be constructed by eliminating aqueous serial dilution of compounds. Traditional serial dilutions that use aqueous diluents can result in errors in dose-response values of up to four orders of magnitude for a significant percentage of a compound library. When DMSO is used as the diluent, the errors are reduced but not eliminated. We use acoustic droplet ejection (ADE) to transfer different volumes of model library compounds, directly creating a concentration gradient series in the receiver assay plate. Sample losses and contamination associated with compound handling are therefore avoided or minimized, particularly in the case of less water-soluble compounds. ADE is particularly well-suited for assay miniaturization, but gradient volume dispensing (also known as “direct dilution”) is not limited to miniaturized applications.
Cost is now a key driver for pharmaceutical companies and in many respects shapes the capital, revenue and resource decisions that have to be made during the drug discovery process. Where companies are resource rich, the need for fully automated screening platforms is reduced and workstation-based systems tend to be more abundant. For companies which are resource limited, fully integrated screening systems tend to provide a good solution but require a high capital investment. As new flexible automation systems are being developed it is now possible to balance the high capital cost of fully automated systems with both revenue and resource savings across wider business needs. This makes fully automated solutions more attractive to big pharma as benefits can be delivered back to the business more quickly.
To facilitate large-scale, high-throughput functional genomics studies using RNAi, we have developed acoustic droplet ejection (ADE) technology to meet the current demands of a successful siRNA library based screen. High-throughput applications require reliable and reproducible transfections to be performed in high density well plate formats. The success of an siRNA experiment relies on the effective delivery of the siRNA molecule. Using ADE technology, a unique tipless and touchless liquid transfer technology used in the Echo® liquid handlers (Labcyte Inc.), we have successfully transfected esophageal cancer cells using a liposome-mediated transfection method. We have also demonstrated the use of ADE technology for reverse transfection. Here, ADE was used to deposit siRNAs in a transfection matrix onto glass slides, then overlaid with a monolayer of adherent cells and the slides incubated to allow reverse transfection. The effects of gene silencing were then assessed by digital image analysis at a single cell level.
Surfactant solutions can be difficult to dispense using high- speed tip- or nozzle-based liquid handling instruments. Low surface tension fluids have a tendency to leak out of tips while bubbles can become entrained or trapped in tips. Both of these mechanisms lead to inaccurate and irreproducible dispensing. Acoustic droplet ejection (ADE; see sidebar below) is a unique liquid transfer technology that eliminates the need for pipette tips, pin tools or nozzles. ADE is proven to deliver more accurate results at low nanoliter volumes of DMSO and salt-based buffers than conventional liquid handling instruments. However, even with ADE, low surface tension fluids containing surfactants can be difficult to transfer accurately and reproducibly. We present here progress towards acoustic droplet ejection parameters for surfactant-containing buffer solutions that deliver volumetric accuracy and precision that is comparable to the established performance for DMSO and salt-based buffer solutions.
The dispensing parameters were evaluated on an automated acoustic liquid handler (Echo® 555, Labcyte Inc.). Triton X-100 was diluted to final concentrations ranging from 0.0001-0.01% in 1x phosphate buffered saline (PBS) containing 0.15 mM sodium fluorescein. ADE was then used to dispense 50 nL of these surfactant-containing solutions from a 384-well Echo qualified acoustic-grade polypropylene source plate (over the entire working volume range of 20 – 50 μL fill volumes) into 384-well destination plates. The destination plates were then filled with 50 μL of 10 mM NaOH and the fluorescent signal was measured from each well on a fluorescence reader.1 Preliminary results yielded average transfer volume precision ranging from 2-5% across all experiments, which is comparable to best-in-class results for DMSO and salt-based buffers.
Creating assay plates in a typical high-throughput research laboratory requires the coordination of several instruments and robotics. Protocol creation processes can be wearisome if the chosen software tool lacks application focus and is simply a hardware “driver”. New assay-plate driven software coupled with a revolutionary scheduler dramatically improves workflow and throughput while eliminating the need for trained software experts. We have designed three software applications specifically to allow end-users to take full advantage of the Labcyte® Echo® liquid handlers and the unique capabilities of acoustic droplet ejection technology. Echo Cherry Pick enables accurate “touchless” low-volume sample transfer from any source well to any assay well. Echo Plate Reformat provides plate replication, reformatting, and region-based mapping tools that facilitate sample pooling into assay plates. Echo Dose-Response automates the “direct” sample dilution process for dose-titration studies. In each of these applications, the user employs an assay plate designer to specify the layout of the assay plates. The software automatically creates the protocol necessary to achieve the desired layout, manages the plates and their contents from protocol start to finish, and generates user-customizable reports in XML or CSV formats for easy import into LIMS systems.
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