featuring the Echo Acoustic Technology
TITLES and AUTHORS
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.
The Echo® liquid handler enables protein crystal formation with sitting drop volumes as little 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 cross-contamination 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 fluid class setting is needed to transfer all crystallography sparse matrix reagents—eliminating the need for multiple calibrations or repeated optimization of fluid class settings.
As biological assays used for HTS and uHTS are miniaturized, it is critical to maintain assay sensitivity while minimizing reagent volumes and maximizing throughput. The GloSensor™ cAMP Assay provides an extremely sensitive and easy to use, real-time luminescent assay format for the interrogation of overexpressed or endogenous GPCRs that signal via changes in the intracellular concentration of cAMP. Tipless, touchless transfers with the Echo® liquid handler eliminate the need for costly disposable tips and greatly simplify assay development efforts. Precise and accurate drop placement eliminates cross-contamination. In this work, we demonstrate optimization of the GloSensor cAMP Assay in low-volume format using a stably transfected HEK293 cell line model expressing the melanocortin 4 receptor (MC4R) and a cAMP-sensing variant of firefly luciferase. We show that the Echo liquid handler is able to titrate small molecule and peptide agonists and antagonists, providing robust assay results that are achieved with significantly reduced volumes of cells and compound.
Miniaturization of assays to low volume 384-well and 1536-well formats has dramatically reduced HTS cost and improved throughput. However, miniaturization has posed some challenges: 1) The difficulty in accurately transferring nanoliter volumes of compound in 100% DMSO into assay plates 2) The low DMSO tolerance of assays (particularly cell-based assays)
This has necessitated an aqueous intermediate dilution of compound prior to addition into assay plates. The effect of this aqueous intermediate dilution on compound solubility has often been questioned.
Recent advances in liquid handling technology has achieved reliable, cost-effective and efficient nanoliter transfer of compound in 100% DMSO into assay plates, improving solubility whilst working within the boundary of DMSO tolerance for the assay. Miniaturization of assays to low volume 384-well and 1536-well formats has dramatically reduced HTS cost and improved throughput.
DMSO, IC50, direct dilution, serial dilution, miniaturization, dose-response
The lifetime and stability of antibody stock solutions, antibody conjugate solutions and enzymes are increased by the addition of cryoprotectants such as glycerol or ethylene glycol. Solutions containing glycerol have historically been problematic to dispense with traditional tip-based liquid handlers. Increased solution viscosity and sample sticking reduces pipetting accuracy and precision, which can be especially significant in nanoliter volume transfers.
The Echo 555 liquid transfer (Labcyte Inc., Sunnyvale, CA) is a completely touchless acoustic transfer technology that can precisely and accurately transfer fluids, including glycerol stock solutions (0-60%). Nanoliter volume transfers were confirmed with a fluorescent marker dye and standard curve for a range of glycerol concentrations. The Echo liquid handler was used to dispense alkaline phosphatase (stored in 50% glycerol) in a traditional phosphatase assay with p-Nitrophenyl phosphate (pNPP) as the substrate with expected activity results. Further experiments showed that a variety of ELISA, kinase, cyclic AMP and cytokine assay kit components can also be transferred using the Echo liquid handler with excellent volumetric accuracy and precision.
The results of these applications illustrate the new capabilities of the Echo liquid handler to enable assay setup, reduce reaction volume resulting in reagent and consumables savings with higher throughput.
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.
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 1536-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 high risk for cross contamination, lower precision, and poor accuracy. The Access workstation relies on the Echo liquid handler and its tipless, touchless transfer of samples and reagents to eliminate such risks—providing high quality assay-ready plates. This poster examines the ability to automate qPCR assay preparation from lysis to analysis while reducing overall reaction volumes to as little as 250 nL.
Miniaturized quantitative PCR (qPCR) in 1536-well plates holds great promise for increased throughput and reagent savings. Delivering reagents to high-density plates can be challenging for conventional tip-based liquid handling systems, leading to inaccurate assay volumes and contamination errors across wells. This study utilized the Labcyte Echo 555 liquid handler to prepare miniaturized qPCR reactions in total reaction volumes ranging from 250 nL to 1.0 µL. The resulting amplification curves yielded excellent crossing point precision and accuracy, with coefficients of variation ranging from 0.95 to 1.25%. Tests with positive and negative controls dispensed into alternating wells revealed zero cross-contamination. These results demonstrate the Echo liquid handler as an ideal platform for preparing miniaturized qPCR reactions in a 1536-well plate format.
Recent innovations in cell lysis reagents and high throughput quantitative PCR miniaturization have allowed researchers to increase throughput while decreasing labor and reagent costs. To ensure high quality data, the liquid handling employed in such low-volume reactions must be precise and accurate. This study utilized the Echo 555 liquid handler (Labcyte Inc., Sunnyvale, CA), the Real-time ready Cell Lysis kit and the LightCycler® 480 system (Roche Applied Science). Cells were grown in tissue culture plates, lysed with the Real-time Lysis solution and then transferred in nanoliter quantities using the Echo liquid handler into 384-well qPCR plates. The plates were then cycled in the LightCycler and the resulting amplification analyzed. Results show excellent standard deviations of less than 0.5 and coefficient of variations of less than 1.5% with volumes as low as 300 nanoliters. The results for this application illustrates the new capabilities of the Echo liquid handler to enable assay setup and reduce reaction volumes resulting in reagent and consumables savings with higher throughput.