Application Notes

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.

real-time PCR, rt-PCR, miniaturization, cross-contamination, "SNP analysis", SNP, app note G102

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.

app note G103

This article describes metabolic profiling applications in which compounds are assayed against several different drug metabolizing enzymes on the same assay plate. We obtain potency data for cytochrome P450 (CYP450) and monoamine oxidase A (MAO) using this parallel approach and testing compounds in a dose-response format using both 384-well and 1536-well plate formats.

cytochrome, bioluminescence, miniaturization, ADME, metabolism
 

Early assessment of ADMET properties (absorption, distribution, metabolism, excretion and toxicity) of drug candidates has become an essential component of modern drug discovery screening to select the overall best scaffolds or leads to the pre-clinical candidate stage. One of the most important ADMET characterizations is the interaction between drug candidates and various human cytochrome P450 enzymes. Cytochrome P450 enzymes are the largest group of drug-metabolizing enzymes and their potential interactions with drug candidates could lead to idiosyncratic toxicity. CYP3A4 is the most abundant cytochrome P450 enzyme in the liver and plays a major role in metabolizing xenobiotics. The Promega P450-Glo™ CYP3A4 Assay (Luciferin-IPA) offers a sensitive, specifi c and high-throughput luminescence assay for the examination of CYP3A4 inhibition. Here, we demonstrate the miniaturization and optimization of the P450-Glo™ Assay with pooled Human Liver Microsomes (HLM) using the Echo 555 liquid handler.

Application Note D100

Early assessment of the ADMET (Absorption, Distribution, Metabolism, Excretion and Toxicity) properties of drug candidates has become a critical part of modern drug discovery. The frequent failure of compounds due to poor ADMET properties necessitates earlier profi ling of ADMET characterizations. As a large number of compounds are screened for ADMET properties against various cytochrome P450 (CYP) enzymes on a regular basis, miniaturization and automation of ADMET assays have become one of the top priorities to manage the cost and labor. The time-dependent CYP inhibition assay is one of the most important ADMET assays which identifies drug candidates that may have potential for undesirable drug-drug interactions or sub-optimal pharmacokinetic properties. Here, we present a miniaturized, simplifi ed and automated method using the Echo 525 liquid handler to evaluate compounds with time-dependent CYP inhibition potential with RapidFire/MS. The Echo 525 liquid handler can precisely and accurately transfer nanoliter droplets completely contact free to avoid any possible contamination. The rapid transfer of various aqueous reagents enables the evaluation of ADMET properties of a large number of compounds. In this application note, we discuss assay results from miniaturized time-dependent inhibition assays. The time-dependent inhibition for two of the most abundant CYP enzymes, CYP3A4 and 1A2, were evaluated. The results from the Echo liquid handler method demonstrate fast, reliable and accurate IC₅₀ values.

Application Note D101

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 quantifi cation curves across 384- and 1536-well plates was excellent with standard deviations less than 0.25 and CVs less than 2.0%. Tests with positive and negative controls dispensed into alternating wells revealed zero cross-contamination. The results demonstrated the Echo 525 liquid handler effectively miniaturized reaction volumes for high-throughput qPCR in both 384- and 1536-well formats.

application note G104

The POD™ automation platform utilizes the revolutionary Echo^® liquid handler and Echo^® software applications to produce low volume assay plates for siRNA screening with unmatched precision and accuracy. Using lower quantities of siRNA and assay reagents makes the POD system the most cost-effective solution for target identification and validation. This highlight reviews the outcome of a study comparing the knock-down of gene expression after siRNA transfection followed by reporter gene and cell viability assays.

Dimethyl sulfoxide (DMSO) is a commonly used solvent for compounds. DMSO accelerates protein unfolding and weakens the binding between small molecules and proteins. Consequently researchers keep DMSO concentrations as low as possible, especially for sensitive assays. To keep the DMSO concentration at less than one percent of the final assay volume has been difficult due to the lack of reliable nanoliter-range liquid handlers. Intermediate aqueous dilution steps can cause the compound to “crash out” of solution. The requirement to keep compounds dissolved while keeping DMSO concentration low in the final assay is especially critical when preparing compound activity curves. The Labcyte Echo™ 550 liquid handler utilizes acoustic drop ejection (ADE) to transfer 2.5-250 nL of compounds in DMSO directly from storage plates to assay plates. Deerac Fluidics Latitude™ bulk dispenser, which uses “spot-on” technology, can precisely deliver as low as 50 nL. The Latitude can be used to rapidly add pure DMSO to specific wells so that all assay wells have the same DMSO concentration. Here we demonstrate the use of these two technologies in combination for keeping final DMSO concentration under 0.5% in HTS assays.

miniaturization, miniaturisation, DMSO, precision, accuracy, linearity, dose-response, assay development and optimization, lead optimization

Early assessment of the ADMET (Absorption, Distribution, Metabolism, Excretion and Toxicity) properties of drug candidates has become a critical part of modern drug discovery. The frequent failure of compounds due to poor ADMET properties necessitates earlier profi ling of ADMET
characterizations. As a large number of compounds are screened for ADMET properties against various cytochrome P450 (CYP) enzymes on a regular basis, miniaturization and automation of ADMET assays have become one of the top priorities to manage the cost and labor. The time-dependent CYP inhibition assay is one of the most important ADMET assays which identifies drug candidates that may have potential for undesirable drug-drug interactions or sub-optimal pharmacokinetic properties. Here, we present a miniaturized, simplifi ed and automated method
using the Echo 555 liquid handler to evaluate compounds with time-dependent CYP inhibition potential with RapidFire/MS. The Echo 555 liquid handler can precisely and accurately transfer nanoliter droplets completely contact free to avoid any possible contamination. The rapid transfer
of various aqueous reagents enables the evaluation of ADMET properties of a large number of compounds. In this application note, we discuss assay results from miniaturized time-dependent inhibition assays. The time-dependent inhibition for two of the most abundant CYP enzymes,
CYP3A4 and 1A2, were evaluated. The results from the Echo liquid handler method demonstrate fast, reliable and accurate IC₅₀ values.

Application Note D102

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.

The Access™ laboratory workstation presents exciting new capabilities for walkaway assay assembly and screening of high-throughput gene expression analysis using qPCR. The Access workstation combines the Echo^® liquid handler, the Roche® RealTime ready Cell Lysis Kit and the Roche LightCycler^® 480 system into a single automated system for gene expression assays with reaction volumes as low as 250 nL. With flexible, wizard-driven software and high quality acoustic liquid transfer, the Access workstation is an ideal platform for both assay development and screening with zero cross contamination.

Cisbio’s HTRF assay technology has a wide range of applications in cell-based and biochemical assays. These assays are readily adaptable to high-throughput screening in 384-well and 1536-well formats. The Echo liquid handler enabled the miniaturization of HTRF assays to 2 μL assay volumes in 1536-well microplates. Reagents and compounds were transferred at 2.5 nL increments, which enabled fl exibility in assay setup and reduced reagent costs. The beta2 adrenergic G-protein-coupled receptor was used as a model target to verify performance of a miniaturized
competitive binding assay and miniaturized cAMP assay.

Application Note C100

Cytochrome P450 (CYP) and monoamine oxidases (MAO) are two groups of enzymes that metabolize drugs. The interactions of these enzymes and compounds are studied carefully in early drug discovery to facilitate greater success in clinical trials. Luminescent metabolism assays consist of the P450-Glo™ Screening Systems (Promega, Madison, WI) for CYP 1A2, 2C9, 3A4, 2C19 and 2D6, as well as the MAO-Glo™ Assay System (Promega) for monoamine oxidase A. A collection of drug-like compounds was profiled by IC₅₀ analyses against a panel of seven (7) metabolism assays in 1536-well format using acoustic droplet ejection (ADE) coupled with low-volume reagent dispensers. ADE was performed with the Labcyte^® Echo™ 550 acoustic liquid handler and low-volume reagent fills were made with a Deerac Fluidics Equator HTS reagent dispenser. ADE and low-volume reagent dispensing provided total enzyme assay volumes of 5 μL. Final analysis volumes were 10 μL.

CYP450, bioluminescence, ADME, Z', "z-prime", dose-response

Miniaturizing quantitative PCR (qPCR) reactions into 1536-well plates holds great promise for increased throughput and reagent savings. However, delivering reagents into such high-density plates can be challenging for conventional tip-based liquid handling systems, leading to inaccurate assay volumes and contamination errors across wells. Labcyte^® Echo 500 series liquid handlers are completely touchless—they use no tips or nozzles, and the dispensing mechanism never touches the reagents in the wells, thereby eliminating well-to-well cross contamination. This study utilized the Echo 555 liquid handler to perform miniaturized qPCR with zero cross contamination, high precision and high accuracy.

real-time PCR, qPCR, rt-PCR, miniaturization, cross-contamination, "SNP analysis", SNP, app note G101

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.

app note G100

The Echo^® liquid handler enables protein crystal formation with sitting drop volumes as small as 50 nL. Nanoliter volumes of protein and crystallography reagents can be transferred into sitting drop wells of crystallography plates. Precise and accurate drop placement eliminates crosscontamination
and ensures drop-on-drop placement for blending protein and crystallography solutions. Precise transfer of reagents ensures repeatable crystallization trials. With Dynamic Fluid Analysis™ only a single fl uid class setting is needed to transfer all crystallography sparse matrix reagents—eliminating the need for multiple calibrations or repeated optimization of fluid class settings. In addition to transferring sparse matrix reagents, the Echo liquid handler can also use Dynamic Fluid Analysis to transfer high concentrations of stock reagents on-the-fly, which allows operators to create optimization grid screens without individually making all variations.

Application Note P100

Cyclic olefin copolymer (COC) is a stiff material of high melting point. Microplates made of COC can be effectively heat-sealed with a laminated seal on a Velocity11 PlateLoc heat sealer.

plate sealing

The POD™ automation platform utilizes the revolutionary Echo^® liquid handler and the Echo^® Dose Response software application to instantly produce higher quality assay plates for dose‐response analysis. With options for integrated plate handling, storage and additional software applications for cherry picking, plate reformatting and more, the POD system provides flexibility to easily create dose‐response curves from samples plated in a variety of formats. This highlight reviews the outcome of a study comparing five separate source plate formats used to generate 12‐point curve assay plates for IC₅₀ determination.