High Throughput RT-qPCR for Identifying Activators of Latent HIV-1 in Primary Human CD4+ T Cells
Labcyte and Roche joint tutorial at SLAS2014
Validation of an Automation Compatible High-throughput Reverse Transcription qPCR Workflow
Labcyte and Roche joint tutorial at SLAS2014
Accelerating Precision Medicine with Efficient Drug Sensitivity Screening
Recently identified associations between variants of cancer genes and drug resistance have increased the value for comprehensive drug sensitivity screening in combination with molecular profiling of cancer cells. In cancer research, the information from drug sensitivity screening is often used to improve the precision of therapy offered to patients. This can involve treatment with repurposed therapeutics, novel therapeutics, or combinations of therapeutics. Comparison of drug sensitivity information along with the molecular profile of certain cancer cells can enable the identification of underlying genetic links to drug resistance.
As these programs are scaled up, operational costs to prepare samples and perform screening can become rate limiting—delaying treatment decisions. Researchers have found that miniaturization from the use of acoustic liquid handling instead of traditional methods has increased the overall efficiency of drug sensitivity screening by lowering costs while improving data quality and throughput. This webinar will review an implementation of the Echo® liquid handler for high-throughput drug sensitivity screening at the Institute for Molecular Medicine in Finland.
During this webinar, Dr. Wennerberg will describe the use of this technology in translational research programs utilizing data from drug. His discussion will include descriptions of the equipment and methods used to efficiently automate drug sensitivity screening, the implementation of acoustic liquid handling technology, and the impact of this technology on screening efficiency and data quality.
What You Will Learn
- How acoustic liquid handling can help drive precision medical treatments based on high-throughput drug sensitivity and resistance screening
- How acoustic liquid handling can generate better data and drive down screening costs
- How the Echo liquid handler can impact applications in genomics, proteomics, and drug discovery
Dispensing Processes Profoundly Impact Biological, Computational and Statistical Analyses
Dispensing and dilution processes may profoundly influence estimates of biological activity of compounds. Published data show Ephrin type-B receptor 4 IC50 values obtained via tip-based serial dilution and dispensing versus acoustic dispensing with direct dilution differ by orders of magnitude with no correlation or ranking of datasets. We generated computational 3D pharmacophores based on data derived by both acoustic and tip-based transfer. The computed pharmacophores differ significantly depending upon dispensing and dilution methods. The acoustic dispensing-derived pharmacophore correctly identified active compounds in a subsequent test set where the tip-based method failed. Data from acoustic dispensing generates a pharmacophore containing two hydrophobic features, one hydrogen bond donor and one hydrogen bond acceptor. This is consistent with X-ray crystallography studies of ligand-protein interactions and automatically generated pharmacophores derived from this structural data. In contrast, the tip-based data suggest a pharmacophore with two hydrogen bond acceptors, one hydrogen bond donor and no hydrophobic features. This pharmacophore is inconsistent with the X-ray crystallographic studies and automatically generated pharmacophores. In short, traditional dispensing processes are another important source of error in high-throughput screening that impacts computational and statistical analyses. These findings have far-reaching implications in biological research.
Miniaturization and Automation of HTRF® Assays with the Echo® Liquid Handler
Cisbio's HTRF assays are amenable to high-throughput screening and miniaturization: the add-and-read format improves ease of use, time and cost savings. Most assays are performed in 384-well and 1536-well formats, at assay volumes of 20 uL and 10 uL, respectively. The Echo liquid handlers from Labcyte use acoustic energy to transfer reagents in a non-contact manner. The low volume increment of the Echo liquid handler enabled an investigation of further miniaturization of HTRF assays to as low as 2 uL. Acoustic transfer capabilities enable not only miniaturized formats but better data quality as tipless transfer prevents loss of hydrophobic compounds to tips and nozzles. Larger screens can be run when used in conjunction with the Access™ workstation. We will show how the Echo liquid handler enables better data quality, better throughput and lower per-well cost.
The Echo 525 Liquid Handler: A New Platform for Accelerated Assay Assembly
The Echo® 525 liquid handler is the newest acoustic liquid handler from Labcyte, enabling acoustic transfer for scientists who run assays from 100 nL to 10 µL. The Echo 525 system transfers biochemical and genomics reagents in 25 nanoliter increments in a non-contact manner--without tips, tubing, or nozzles. Dynamic Fluid Analysis™, a unique processing feature developed by Labcyte, ensures that scientists do not have to stop and calibrate their instruments before changing reagents or transfer volumes. This increases the flexibility to perform assay development and enables complete assembly of efficient microliter-scale assays. We will demonstrate a few ways in which the Echo 525 liquid handler can simplify processes in biochemical and genomic assay setup.
A New Approach to Early ADME CYP450 Inhibition Profiling Using the Echo® Liquid Handler
The high cost of drug discovery and the frequent failure of compounds due to poor ADMET (Absorption, Distribution, Metabolism, Excretion and Toxicity) properties necessitate an earlier study of ADMET profiles. Efficient early profile determination is an expensive process unless the process is miniaturized, and the cost per data point is reduced. The CYP450 inhibition assay is one of the most important ADMET assays which identifies drug candidates that may have undesirable drug-drug interactions or sub-optimal pharmacokinetic properties. Here, we present two miniaturized, simplified and automated methods using the Echo® liquid handler to evaluate CYP450 inhibition and time-dependent inhibition potential. When utilized in tandem with an integrated microplate reader or with RapidFire/MS, the new methods generate high quality IC50 data while reducing reagent costs by up to 90%.
Advances in High-Throughput One-Step qPCR Directly From Cells Enabled by the Echo® Liquid Handler and Roche LightCycler®
Quantitative reverse transcriptase PCR (RT-qPCR) offers unmatched accuracy and quantification of gene expression. The Roche LightCycler 1536 offers a unique solution for preserving proven qPCR assay formats while significantly increasing throughput over 384-well instruments. While providing increased throughput, upstream workflows for generating template for qPCR assays through RNA isolation and subsequent cDNA synthesis can be the rate limiting step for high-throughput qPCR set up. Combining a one step RT-qPCR assay with a streamlined workflow for generation of template from cellular material, higher density 1536-well microplates bring high throughput analysis while employing fewer reagents. To successfully miniaturize RT-qPCR assay preparation for high-throughput workflows, a liquid handler must be able to dispense nL volumes with high precision and accuracy while avoiding cross contamination. The Echo liquid handler overcomes these challenges using tipless, touchless, acoustic transfer of reagents to deliver the precision and accuracy needed for high quality qPCR. The Access™ workstation combines the benefits of the Echo liquid handler and novel reagent chemistries with qPCR analysis into a single walk-away platform for high throughput qPCR. This session examines the ability to streamline qPCR assay preparation from generation of template to analysis utilizing the RealTime ready Cell Lysis Kit and LightCycler 1536 system from Roche on the Access workstation from Labcyte—increasing throughput and reducing overall reaction volumes to as little as 500 nL.
Advances in Acoustic Protein Crystallography Screening Using the Echo® Liquid Handler
The diverse array of aqueous solutions inherent to protein crystallography screening presents a challenging venue for acoustic-based liquid transfer, but offers compelling advantages in ease of use, small crystallization volumes, and thereby significant protein reagent savings. I share our experiences with acoustic setup of successful crystallization trials using 10-30 nL protein per droplet (both advantages and limitations), technical advances in the Echo system that yield improved performance, and methods for implementing a rapid and streamlined nano-scale crystallization workflow.
High Throughput Screening with siRNA Libraries—Optimizing Assay Performance to Improve Data Quality
The use of siRNA technology to probe the details of gene function in cells has proven to be a powerful research tool. Recently, siRNA technology has expanded into HTS applications such as the screening of whole genome siRNA libraries. There have been many technical difficulties adapting siRNA assays to HTS platforms and work continues in this area.
The HTS Center at Southern Research Institute has a long history and depth of experience with small molecule screening in a wide range of assay types. With this background we have established metrics on assay performance that are rigorous and produce excellent results for drug development. When we started to develop siRNA screening technology, we expected to achieve the same assay metrics.
Over the last year, a number of technical problems were identified and resolved in pursuit of this goal. As a result of this work, we now have siRNA assays with Z values of 0.7 and higher, CVs of less than 10% and a high degree of reproducibility using either Pearson's or Spearman's Correlations.
Many factors contribute to the production of high quality siRNA data including equipment selection and methodology. The technical aspects of how this was achieved will be discussed, including how some of the more challenging problems were addressed and why specific pieces of equipment were selected for each step in the process.