Assay Assembly for Miniaturized Quantitative PCR in a 384-well Format Using the Echo® Liquid Handler
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
Automated RT-qPCR Utilizing the Access™ Workstation, the Echo® Liquid Handler, and RealTime Ready™ Reagents from Roche Applied Science
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
Bioluminescence for Drug Metabolism Profiling
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
Filling MicroClime® Environmental Lids
The Labcyte® MicroClime environmental lid minimizes edge effects and preserves the concentration of solutions in microplate wells. Unlike standard microplate lids, the MicroClime lid incorporates a unique, fluid-absorbing matrix. The absorbed fluid creates a vapor barrier that protects the sample from the exterior environment, especially near the edges of the microplate. The MicroClime lid is easy to use—simply place it on or lift it off a microplate. No specialized tools are required, and it is compatible with robotic plate lidding systems. The lid can be filled with many different fluids, using either a hand-held syringe or automated bulk fillers.
When filled with anhydrous DMSO, the MicroClime lid prevents evaporation and hydration of DMSO in compound library plates during storage. It also stops evaporation of ultra-low-volume DMSO droplets in screening assays. The lid can also be filled with water to prevent evaporation in aqueous assays.
High Throughput Miniaturization of Cytochrome P450 Inhibition Screening Using the Echo® Liquid Handler
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
High Throughput RT-qPCR for Gene Expression Analysis
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.
High-throughput Miniaturization of Cytochrome P450 Time-dependent Inhibition Screening Using the Echo® 525 Liquid Handler
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
High-Throughput, Low Volume, siRNA Screening
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.
Keeping DMSO Concentration Below 0.5% to Minimize its Effect in HTS 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
MicroClime Environmental Lids Reduce Edge Effects in Multi-Well Plates
Edge effects—the tendency of the contents of wells in the outer rows and columns of a multi-well microplate to vary from wells in the interior of the plate—are due to interactions of the contents of the wells of the microplate with the laboratory or storage environment. Evaporation is rapid at the corners and edges while wells near the center are protected from change. Hygroscopic liquids (e.g., dimethyl sulfoxide (DMSO)) can rapidly absorb water from the environment and the tendency towards hydration is greatest at the edges and corners. The gradient between the contents of a plate and the environment is greatest at the edges of the plate and the fastest changes occur at that steep gradient.
Miniaturization of Cytochrome P450 Time-dependent Inhibition Screening Using the Echo® Liquid Handler
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
Miniaturization of Multiple Displacement Amplification (MDA) of Genomes from a Single Cell
Advances in whole-genome amplification have enabled sequencing of DNA and RNA from a single cell, or what is commonly called Single-cell Genomics (SCG). SCG has become an invaluable approach to understanding the magnitude of genetic and epigenetic variation that occurs throughout the life of an organism. Despite the growing interest in SCG, a standardized method for sample preparation currently does not exist. However in a recent publication of Nature Protocols, researchers at the Joint Genome Institute describe an approach that can meet the throughput and sample quality required for robust whole genome amplification from a single cell. This application highlight, describes the role of the Echo® liquid handler in this new approach.
Application highlight G106
Miniaturization of the KASP™ Genotyping Assay for Maize with the Echo® 525 Liquid Handler
Single nucleotide polymorphism (SNP) genotyping of maize samples is commonly performed in agricultural science to aide marker assisted selection, study heterosis and a variety of other biological behaviors. Its widespread adoption in agricultural science has been challenged with increasing reagent costs and labor intensive multi-step processes. Acoustic non-contact liquid handling using the Echo 525 liquid handler offers unique advantages to traditional processes by incorporating a tip-less solution to deliver reagents precisely and accurately. Assay miniaturization is enabled with high accuracy and precision at volumes as low as 25 nL. This study utilized the Echo 525 liquid handler to miniaturize a KASP genotyping assay for maize at a throughput meeting the demands of most high throughput production processes. The results demonstrate miniaturization with the Echo 525 liquid handler without compromising throughput targets.
Miniaturized AlphaLISA® Assays Using the Echo® Liquid Handler
Labcyte Echo liquid handlers transfer compounds and reagents at nanoliter volumes without the use of tips or contact. This capability provides the precision and accuracy to miniaturize biochemical and cell-based assays for screening in 1536-well microplates. AlphaLISA technology is a faster, no-wash alternative to traditional ELISA assays for biomarker and cytokine detection. With high sensitivity and low background, these assays can be readily adapted to miniaturization with the Echo liquid handler. To complement the performance of the Echo liquid handler, the BMG PHERAstar® FS plate reader provides excellent sensitivity and throughput for miniaturized, high density assays. In this application note, we use a TNF-α AlphaLISA assay to validate the performance of a miniaturized assay.
Application note D104
Miniaturized and High-Throughput Metabolic Stability Assay Enabled by the Echo® Liquid Handler
Early assessment of ADMET (Absorption, Distribution, Metabolism, Excretion and Toxicity) properties plays a critical role in the optimization and prediction of pharmacokinetic behaviors of new chemical entities. Among the most important ADME assays is the in vitro drug metabolic stability
assay. This assay evaluates the susceptibility of compounds to biotransformation or intrinsic clearance. Because an increasing number of compounds require metabolic stability evaluation, a high-throughput and cost-effective method is desirable during early drug discovery. In this application note, we discuss how the Echo liquid handler provides a platform for conducting a miniaturized high-throughput metabolic stability assay. The Echo liquid handler precisely and accurately transfers nanoliter volumes with acoustic energy in a completely contact-free manner. Thus high quality data can be obtained with a small fraction of the volume required by tip-based liquid handling methods.
Application note D103
Miniaturized Drug Sensitivity and Resistance Testing with the Echo® Liquid Handler and Access™ Workstation
Recently identified associations between variants of cancer genes and drug resistance increase the value for comprehensive drug sensitivity and resistance testing in combination with molecular profiling of cancer cells. The measure of sample sensitivity or resistance to a drug requires high throughput screening of engineered cancer cell lines or samples directly from affected patients against combinations of anti-cancer therapeutics. Results are compared with genetic profiles in an attempt to determine the more effective treatment. Advancements in next generation sequencing and qPCR technologies persuade many research organizations to increase effort in these areas. In doing so, researchers immediately recognize screening efficiency as a critical factor to accurate and reproducible drug sensitivity and resistance testing. This application note discusses the implementation of miniaturized drug sensitivity and resistance testing, at the Institute of Molecular Medicine in Finland (FIMM), with assay-ready plates produced by the Echo liquid handler.
Application Note C101
Miniaturized Enzymatic Assays with Glycerol
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.
Miniaturized Gene Expression in as Little as 250 nL
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.
Miniaturized HTRF® Assays Using the Echo® Liquid Handler
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
Miniaturized Luminescent Metabolism Profiling Assays
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