featuring the Echo Acoustic Technology
TITLES and AUTHORS
Quantitative PCR (qPCR) is a prevalent tool spanning many phases of drug discovery. Advances in qPCR detection to enable 384- and 1536-well microplate formats have incentivized researchers to miniaturize qPCR assays as a means to offset the costs of increasing throughput. To significantly reduce qPCR volumes and maintain data quality, the liquid handling methods employed for such low-volume transfers must be precise and accurate. Tipless, touchless acoustic droplet ejection with the Echo® liquid handler eliminates the cost of disposable tips or tip-wash cycles and simplifies assay setup by eliminating dilution steps. This study utilized the Echo 525 liquid handler to assemble low-volume qPCR assays at speeds that keep pace with high-throughput demands. Precision for the resulting quantification curves across 384- and 1536-well plates was excellent with standard deviations less than 0.25 and CVs less than 2.0%. The results confirm the advantages of using the Echo 525 liquid handler to miniaturize reaction volumes for high-throughput qPCR in both 384- and 1536-well formats.
Despite the growing value of RT-qPCR to quantify mRNA expression levels, large-scale application of the technique has been hindered by laborious multi-step processes with high reagent costs. Recent advances in reagent technology, such as the RealTime ready cell lysis reagents from Roche Applied Science, simplify the path from sample preparation to analysis. Utilizing these reagents in a miniaturized and automated workflow reduces overall operating costs and warrants the use of RT-qPCR on a larger scale. This study discusses the implementation and validation of a miniaturized and automated workflow with the Access™ workstation for high-throughput RT-qPCR. The validation of the individual steps and the overall workflow are presented.
The need to transfer fluids in the 1-10 μL volume range is an important concern when assembling biochemical assays. Liquid handlers capable of dispensing fluid volumes in the 1-10 μL volume range typically use pipette tips which are disadvantageous due to tip costs, tip cleaning requirements and the possibility of contamination. For researchers in the genomics and proteomics fields, the ability to accurately and precisely dispense all reagents in order to build a completebiochemical assay in the 1-10 μL volume range is highly beneficial.This work highlights utilizing acoustic droplet ejection (ADE), a liquid transfer technology platform that eliminates the use of tips or nozzles and has been proven to accurately and precisely transfer fluids in the low-nanoliter volume range. Here we introduce the new Echo 525® liquid handling platform, which utilizes ADE technology to accurately and precisely dispense aqueous buffered solutions at 25 nL increments. The volumetric flow rate is approximately 5 μL/s. Both accuracy and precision of dispensing at this volume and flow rate is comparable to that ability of a few general liquid classes to accurately and precisely transfer various aqueous reagents without requiring individual calibrations.
New biologicals such as venoms have emerged as key tools in disease research for increased specificity and potency. Venoms have already shown great utility in disease areas such as cancer, pain, haematology, antimicrobials, immunology and natriuresis. The scientific literature in this arena has exhibited rapid growth with the use of venom components as novel leads and therapeutics for targets such as ion channels, enzymes, GPCRs, growth factors and others1-6. This work demonstrates acoustic liquid handling of key components of a Venom Discovery Array™ (VDA) stored in an acoustically qualified 384-well plate which enables researchers to safely handle and accurately transfer venoms.
The broad range of venom fluid properties requires a key feature enabled on the Echo® liquid handler termed - Dynamic Fluid Analysis™ (DFA) which adjusts power and acoustic focus in real time on a well-by-well basis enabling precise and accurate transfer from an Echo qualified source plate. By compensating for inherent variations in potent, biological fluids such as venoms, the Echo liquid handler can dispense components of the Acoustic Venom Discovery Array™ as it would a compound library or a natural products library stored in DMSO. For this work, neat Naja nigricollis (black-necked spitting cobra) venom was diluted in 1 x PBS and transferred in a dilution series from 65% - 20% (v/v) with excellent accuracy and precision (both < 8%). The venom transfer is non-contact and tipless, with increased cost savings from elimination of tip costs and washing fluids.
The current standard of accuracy for quantification of gene expression is reverse transcriptase quantitative PCR (RT-qPCR).1 The method offers the highest level of precision, specificity, and sensitivity. Specialized instrumentation and kits manufactured by life science companies have simplified RT-qPCR protocols to the point that it is now the most straightforward and reliable investigative tool used in a multitude of laboratories performing gene expression. Researchers frequently use RT-qPCR to investigate cellular mRNA fluctuation in response to experimental conditions, such as treatment with small molecule compounds, protein therapeutic candidates, and foreign RNA. While RT-qPCR is a valuable experimental technique, it is traditionally regarded as a low throughput method by comparison to other means of quantifying gene expression. Reasons for this include the number of steps required to produce resulting data, the amount of sample required, and the relatively high cost of reagents. As a result, higher throughput and more cost-effective assays are performed first. Once interesting experimental conditions are identified, RT-qPCR is used to validate lower-cost, higher throughput methods.
Recent improvements to RT-qPCR sample preparation reagents and protocols, combined with existing non-contact, nanoscale liquid handling technologies enable RT-qPCR assay automation, miniaturization, and optimization. Now, this “gold standard” of quantitative gene expression analysis can be used to improve productivity and efficiency by providing accurate gene expression data in earlier screening studies. A new, cost-effective sample preparation method for RT-PCR incorporates a “one-step” cell lysis buffer which eliminates the need for mRNA isolation and purification.2 This improved method, when combined with nanoscale non-contact liquid handling3 technology enables a high throughput automated workflow for gene expression.
The Echo® liquid handlers incorporate Dynamic Fluid Analysis™ to optimize acoustic transfer on-the-fly. Starting with a default setting for a broad fluid class, each transfer is further optimized in real-time to account for various changes in fluid properties. This enables accurate and precise transfer of reagent sets that may contain surfactants or glycerol, which would require multiple calibrations on traditional liquid handlers. This application highlight discusses the capability of the Echo liquid handlers to transfer low volumes of enzyme stored in 50% (v/v) glycerol into 384-well plates. This allows both the simplification of protocols by removal of an intermediate dilution step, and improved performance in a miniaturized format.
Quantitative reverse transcriptase PCR (RT-qPCR) offers unmatched accuracy and quantification of gene expression. Combining the one step RT-qPCR assay with a streamlined workflow upstream for generation of template from cellular material has enabled the use of higher density 384- and 1536-well microplates in high throughput methods employing fewer reagents. To successfully miniaturize RT-qPCR assay preparation for high-throughput workflows, one must overcome high risks for cross contamination, lower precision, and poor accuracy. The Access workstation combines the benefits of the Echo® liquid handler with novel reagent chemistries and qPCR analysis into a single walk-away platform for high throughput qPCR. The Echo liquid handler overcomes the challenges of assay miniaturization by enabling tipless, touchless transfer of reagents to deliver the precision and accuracy needed for high quality qPCR. This poster examines the ability to automate qPCR assay preparation from lysis to analysis utilizing the RealTime ready Cell Lysis Kit and LightCycler® 1536 system from Roche on the Access workstation—increasing throughput and reducing overall reaction volumes to as little as 500 nL.
Small scale protein crystallography offers tremendous advantages in screening conditions against small quantities of protein. A drawback of traditional liquid handling capabilities is reproducibility at small volumes; since reagents vary in viscosity, surface tension and other fluid properties, it is often challenging to validate and repeat small-scale reagent transfer. Acoustic transfer with the Echo liquid handler simplifies the small scale crystallography process by transferring reagents precisely at 2.5 nL increments. The Echo liquid handler can adjust fluid transfer parameters on the fly on a well-by-well basis. This allows a single, universal fluid type to be used to transfer a wide range of viscous and osmotic fluids, simplifying the liquid transfer process. We demonstrate the precision of transfer for a wide variety of concentrated reagents common to protein crystallography sparse matrix screens, and demonstrate reproducibility in the protein crystallography process as a function of acoustic transfer of reagents.
RT-qPCR has been increasingly utilized by researchers to quantify mRNA levels. Its widespread use has been limited by an often laborious multi-step process and high reagent cost. Recent advances in non-contact reagent dispensing and next generation one step lysis reagents are enabling scientists to generate high throughput data at a much faster rate. This technical note describes the automation of three complementary tools to generate high throughout RT-qPCR data; the Echo liquid handler, RealTime Ready cell lysis reagents, and the Access workstation.
Early assessment of cytochrome P450 (CYP) inhibition has become an essential component of drug discovery screening. The CYP3A4 enzyme is the most abundant enzyme in the liver and plays a major role in metabolizing many xenobiotics in humans. The Promega P450-Glo™ CYP3A4 Assay (Luciferin-IPA) offers an extremely sensitive, high throughput and specific luminescence assay for the examination of CYP3A4 inhibition with pooled Human Liver Microsomes. The Echo 555 liquid handler provides precise and accurate acoustic transfer free of cross-contamination risk. Here, we demonstrate optimization of the P450-Glo™ Assay with Human Liver Microsomes (HLM) in low volume format using the Echo 555 liquid handler. We show that the Echo 555 liquid handler can transfer microsomes, inhibitors and substrate in nanoliter volumes, providing a sensitive and robust small scale assay. IC50 results for four known CYP3A4 inhibitors using our miniaturized assay compared favorably to previously reported values in the literature.
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