The HTS laboratory provides the following services to Cancer Center researchers:
- Target assessment and assay evaluation
- HTS assay development and testing
- HTS screening of chemical compounds or siRNA libraries
- Data analysis and storage
- “Hit” validation
- Compound libraries
Cancer Center Support for HTS Experiments
The cost of an HTS experiment is substantial. For approved projects, the Simmons Cancer Center HTS core laboratory will provide access to the UTSW compound collection and/or to genomic siRNA resources. The core will provide the following without charge:
- Compounds or siRNAs
- Access to equipment needed for HTS
- Aid in conducting HTS experiments
- Data processing and analysis
Laboratories conducting HTS experiments must dedicate at least one scientist to conduct the experiments and must provide all reagents needed. In addition, there are several types of fees that may be charged to recover reagent costs associated with preparing compound or siRNA assay plates from library source plates. The HTS Core assists investigators in identifying reagent sources. The Simmons Cancer Center will cover one half of the reagent costs for approved projects to be conducted by Cancer Center Members.
Both the practicality and the scientific and therapeutic value of targets for screening are assessed by the Cancer Center HTS Oversight Committee. The HTS Core staff will then assess the cost and practicality of the proposed assay for HTS. This includes aspects such as the number of liquid handling and wash steps, timing, reagent volumes, and necessary controls.
For advice about these aspects in preparation for applying for Cancer Center Support, contact Michael Roth, Ph.D.
HTS staff work with the initiating laboratory to develop the proposed assay into a form suitable for HTS. Compound screens are run in 384 well plates without replicates. siRNA experiments are typically run in triplicate in 96 well plates. For cost and reliability reasons, assays must be end-point assays and simplified to minimize liquid handling steps.
The assay signal-to-noise ratio must be optimized to produce statistics indicating that a compound or siRNA with the desired activity can be reliably distinguished from compounds or siRNAs lacking activity in the assay. No experiment will have access to compound or genomic siRNA resources unless the assay reproducibly achieves the desired signal-to-noise statistics.
HTS experiments require controls on each plate both for quality control and for data normalization. Both positive and negative controls must be optimized. The HTS laboratory works to minimize the cost of the experiment, which often requires changing reagents and sometimes completely redesigning the assay.
Once an assay has an acceptable signal-to-noise ratio in small-scale experiments, it is tested in 10 to 20 plates treated with DMSO to determine reproducibility under conditions similar to HTS screening. Assays that are reproducible are then screened against a test library of 8,000 compounds at 5 µM concentration to determine the “hit” rate.
Assays that pass these evaluations are then screened at rates between 8,000 to 19,000 compounds per day, depending upon the assay type. Cancer Center members are required to supply a scientist to conduct the HTS screen. This person will be aided by the HTS staff, who operate the robotic liquid handlers and perform all operations involving the dispensing of compounds from library plates to experimental plates.
siRNA experiments are developed in a similar manner, except that they are run in 96 well format in replicate plates and no test library is used before the genomic screen is begun. Just as with compounds, siRNA experimental controls must achieve a reproducible signal to background ratio in runs of multiple plates before the project is allowed to screen the genomic library.
Only complete genomic screens of the siRNA library are allowed.
HTS experiments generate a large data set in which the link between well location, content, and experimental value must be rigorously maintained. HTS staff are responsible for this, as well as for monitoring daily quality control and flagging plates with unacceptable control statistics that must be repeated. The HTS Core processes screening data and adds it to the Laboratory’s screening database, which connects compound or siRNA identity to activity in each assay run by the lab.
The HTS laboratory provides screening data in Excel files to the initiating investigators and, at the conclusion of the primary HTS experiment, aids in defining compounds or siRNAs of interest.
The HTS laboratory assembles a “hit collection” of compounds or siRNAs that had activity in the assay exceeding a predetermined value, typically three standard deviations or more from the mean of the experimental population. The “hit collection” is screened again in the primary assay and compounds or siRNAs that show repeated activity are then subjected to additional secondary assays that can be run in multi-well format with the limited amount of compound or siRNA available.
Based upon the results of secondary screens, lack of activity in other HTS experiments run by the Core (screening history) and other criteria such as compound structure, a small number of compounds or siRNA oligonucleotides are purchased for continued investigation in the initiating laboratory. This is a critically important step in the HTS process. Because subsequent experiments are time consuming and expensive, very few compounds or siRNAs can be subjected to more intense analysis.
The typical compound set chosen for follow-up is 10 to 20 from an initial hit collection of several thousand. For siRNA experiments, one can choose to select siRNAs of interest out of a second genomic library at a cost of $5,000 for each experiment.
Once compounds of interest are purchased for additional experimentation, the purity and identity of the compounds can be determined by the Medicinal Chemistry Core in the Biochemistry Department on a fee for service basis (contact Noelle Williams, Ph.D. at email@example.com). In cases where compounds are no longer available commercially, commercial suppliers will usually contract to synthesize the compounds. The Medicinal Chemistry core has the capability to synthesize a limited number of compounds each year.
The HTS Core laboratory has multiple copies of 200,000 small, drug-like compounds dissolved in DMSO and arrayed in 384 well plates. The compounds were purchased from major suppliers to the pharmaceutical industry with a small but growing number of compounds synthesized at UT Southwestern. Compounds were selected for being able to pass 48 structure-based filters that identified undesirable characteristics as well as for satisfying a relaxed set of Lipinski’s' rules for good bioavailability. Those that passed and were purchased represented the desirable structural diversity available from the companies shown below as of November 2004 (Comgenex, 2005).
Compounds in the UTSW library were purchased from ChemDiv (100,000), ChemBridge (75,500), ComGenex (22,000), Prestwick, (1,100) & TimTek (500).
Genomic siRNA Libraries
The HTS core manages a human genomic library from Dharmacon and Drosophila genomic RNAi library from Ambion that were purchased by UT Southwestern and are available free of charge to UT Southwestern investigators who propose experiments acceptable to an Oversight Committee (contact Michael Roth, Ph.D. at firstname.lastname@example.org for information). The human genomic library is composed of pools of four double-stranded siRNA oligos specific for each of 21,125 human genes arrayed in 96 well plates.
The HTS Core laboratory also manages smaller quantities of a similar human genomic library purchased from QIAGEN by the NCI program project led by Steve McKnight, Ph.D. This second library is used to assemble smaller sets of siRNAs to validate hits from primary screens of the Dharmacon library. It is available to Cancer Center investigators outside the PPG at a cost of $5,000 per experiment.
In addition to being composed of pools of siRNA sequences for each gene distinct from those comprising the Dharmacon library, the QIAGEN library is the only cost effective way to validate a collection of several hundred potential hits resulting from a genomic screen.
Currently individual pools of siRNAs targeting a gene cost about $100 per pool from either Dharmacon or QIAGEN, making validation of the entire set of “hits” from a genomic screen prohibitively expensive if they are purchased individually. Use this link for information on ordering oligos from Dharmacon. All libraries are kept in multiple copies in -80 °C or -20°C freezers within the 1,900 square foot HTS laboratory.