Overview and Guidelines

Overview and Guidelines

“What Every New Screener Should Know” Introduction to the Screening Process

Click here for a link to view or download the ‘What Every New Screener Should Know’ document, which is an important introduction to screening at the ICCB-Longwood facility.  This is a quick reference guide that is distinct from the additional information below.

 

Initiating a Screening Project at ICCB-Longwood

The ICCB-Longwood Screening Facility collaborates with academic investigators to perform high-throughput screens of chemical and RNAi libraries. Our compound collection includes over 500,000 small molecules, including commercially available compound libraries and unique natural product libraries. We also offer genome-scale RNAi screening. Dharmacon SMARTpool siRNA libraries targeting the whole human genome (18,220 genes), the whole mouse genome (20,552 genes) and 3 different human miRNA collections are available for screening at ICCB-L. Data generated from screens are entered into a screening results database. Access to this non-public database (Screensaver) is granted only to ICCB-Longwood investigators and collaborators who have deposited data. For more information on small molecule and RNAi data in the database, please see the ICCB-L small molecule user agreement and ICCB-L RNAi user agreement, both area available to download on the Screening Documents page.

ICCB-Longwood screening facility personnel assist efforts by providing access to the compound and RNAi libraries, and by operating and maintaining the screening robots. They also offer advice at all stages of the screening process, including assay design and optimization, screening, data analysis and follow-up approaches.  Collaborators perform their own assays and provide their own supplies, including assay plates. A fee is charged for access to screening at ICCB-Longwood. Please see the small molecule application page and the RNAi application page for more details about the fee structures.

To allow us to prioritize new screening projects appropriately, a formal application must be submitted in order to initiate a screen at ICCB-Longwood. Additional applications must be submitted each time a new screen is proposed. We currently review applications on a rolling basis (links to the applications appear in the previous paragraph or on the left hand navigator bar under Application Forms).

To avoid situations in which two different groups are carrying out substantially similar screens on the same small molecule collections or RNAi libraries at the same time, we attempt not to initiate screens that compete with existing ones. Whether a proposed screen will compete with ongoing assays is decided by the ICCB-Longwood Screening Application Review Committee in consultation with the investigators involved. Precedence is generally given to the screen first proposed.

Radioactive assays are currently not supported in the ICCB-Longwood Screening Facility, and substances requiring a higher containment level than BSL2 are not permitted.

Please send questions about the application process to screeniccb_apply”AT”hms.harvard.edu.

After an application is accepted, the investigator is granted access to the screening facility. Click to link to the document “What Every New Screener Should Know”, which is requisite reading before a screener’s first visit to the ICCB-Longwood Screening Facility.

 

Biosafety Requirements

The maximum biocontainment level for studies conducted in the screening facility is BSL2.

Biosafety procedures for the screening facility are governed by Harvard Medical School regulations, overseen by the Committee on Microbiological Safety (COMS). The screening facility currently holds a biosafety approval covering a variety of organisms.  There may be experimental design constraints for certain organisms that are considered highly infectious or that present an unusual health hazard.

Biosafety requirements are evaluated when an equipment training request is submitted.  If you are wondering whether the organism(s) you would like to work with has been approved for use at ICCB-Longwood, please contact Jennifer Smith (jennifer_smith”at”hms.harvard.edu) or Katrina Rudnicki (katrina_rudnicki”at”hms.harvard.edu).  If your screen involves items not approved, we can file an amendment to add your reagents to the list. To assist us in this process, if you are not a Harvard Medical School-affiliated researcher, please provide a copy of your institutional biosafety committee approval for all research procedures applicable to the screen. Depending on the format of your institutional approval, you may have to provide additional information describing your proposed work. For example, the COMS application requests names and descriptions of strains, plasmids, experimental design, and biosafety issues. Please review the HMS COMS application form to see what is required. Please be aware that COMS approval may take a month or more, so it is important to file amendments as early as possible.

Your BSL2, hazardous waste, and bloodborne pathogen training should also be completed and up-to-date. Annual refresher courses for Harvard personnel are available online through Environmental Health and Safety.

For screeners not holding current Harvard IDs, access to the screening facility is contingent upon your institutional biosafety office co-signing the screening facility user agreement to indicate that your employer has provided all necessary BSL2, hazardous waste, and bloodborne pathogen training and protective equipment. For practical purposes, however, site-specific training and protective equipment will be provided by the screening facility as necessary.

All ICCB-L screeners must complete the ICCB-L Screening Facility Lab Specific Training prior to beginning any work in the facility.  This brief training reviews the location of emergency equipment and policies specific to the screening rooms.  Please complete the online Training Request Form to get started. In addition, the ICCB-L Lab Safety Handbook can be viewed or printed here (PDF).

 

Designing a High Throughput Assay

Assay Volume and Plate Type

For a high-throughput screen, a biochemical or cell-based assay must be adapted to microtiter plate format. Assays are most commonly performed in 384-well assay plates; in certain cases, they may also be performed in 96-well or 1536-well plates. Assay volumes in 384-well plates range from 5 µL (in low-volume plates) to 100 µL (in standard plates). At the low end of this range, inaccuracies in small-volume pipetting can cause signal variation, while there is a risk of spillage and cross-contamination at the high end. We therefore recommend that, if possible, investigators use 30-50 µl assays in standard plates  and avoid dispense volumes lower than 5 µL.

Different types of plates are recommended depending on the assay detection method used. White plates are recommended for luminescence assays, black plates for fluorescence assays, clear-bottom plates for automated microscopy, and clear polystyrene plates for photometric assays. Please see the Screening Supplies section for standard plate options.

Replicates and Controls

Most high-throughput assays show a high amount of inherent variability and error. Therefore, it is strongly recommended that small molecule assays be performed in duplicate and RNAi assays be performed in triplicate. Using replicate data points can reduce false positive rates by up to one-half.

Control readings are essential to a well-designed assay, and every assay should use as many controls as possible. In general, there are two types of controls: plate-based controls and assay-wide controls. Plate-based controls are controls that are placed on each individual assay plate. These are essential for identifying plate-to-plate variability and establishing assay background levels. Assays that are prone to plate-based variability (such as luciferase readouts that decay over time) should primarily use plate-based controls and normalization. Stock library plates are formatted with empty wells to allow for screen-specific plate-based controls. It is good practice to use all available wells, with the researcher deciding on the appropriate distribution of positive and negative controls.

For small molecule screens, the ideal positive control is a small molecule that is known to produce the sought-after effect. Negative controls should match experimental conditions and only lack compound. For RNAi screens, the positive control should be an siRNA that knocks down a gene in the pathway of interest, producing the desired phonotype, while the negative control should be an identified non-targeting siRNA.

Assay-wide controls are separate plates containing only control wells and no screening compounds. These are particularly useful for determining the background levels of an assay and should be used to help determine whether an assay has sufficient signal to be reliably detected.

Signal/background ratio (S/B) and well-to-well variability (CV) are important issues to consider. As assay variability increases, the S/B ratio must increase for the screen to be successful. We recommend using a positive control condition to determine the S/B ratio. To determine S/B, fill a plate with reagents using the same equipment to be used for the screen. Add several dilutions of the positive control to several wells, and determine whether the positive control can be reproducibly detected above the well-to-well variation. These data will provide an indication of the false-positive and false-negative rate of your assay.

Quantitative Assay Evaluation and Optimization

The Z´ factor calculation is useful during piloting for quality assessment of assay conditions (Zhang et. al. 1999, see below for full reference). An assay can be considered validated for high-throughput screening after independent experiments (for example, each experiment set up separately starting from scratch or carried out on different days) have been shown to result in reproducible and suitable Z´ factor values. Each experiment should be performed on at least one full 384-well plate on which ½ of the wells contain positive controls and ½ of the wells contain negative controls. This will produce a statistically significant data set for evaluation. The experimental conditions to be used during the screen, including use of robotics, should be mimicked to the greatest extent possible.

To quantitatively rank assay conditions, perform control experiments and calculate Z´ factors from the data collected:

SD + = positive control standard deviation

SD – = negative control standard deviation

Ave + = positive control average

Ave – = negative control average

For small molecule screening assays, the below Z’ values indicate assay fitness:

1 > Z´ > 0.9 An extremely robust assay

0.9 > Z´ > 0.7 A robust assay

0.7 > Z´ > 0.5 Acceptable, but identification of positives will benefit significantly from any improvement

0.5 = Z´ The minimum recommend for high throughput small molecule screening

This table may differ slightly from published recommendations. However, it is based on the general experience of small molecule screeners at ICCB-L. We commonly observe that screening results rarely achieve the high quality levels seen during piloting using controls. Also, cell-based assays frequently cannot achieve as high a Z’ factor as assays using purified proteins.

For RNAi screening assays, the experience of ICCB-L screeners has been that Z’ factor values under the most optimized conditions are often less than 0.5. These RNAi screening assays have nonetheless been productive.

Please contact us at screeniccb_apply@hms.harvard.edu if you require more information.

Z’-factor reference: Ji-Hu Zhang, Thomas D. Y. Chung and Kevin R. Oldenburg (1999). A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen 4:67-73.

 

Performing a Screen

Once a satisfactory assay is developed in 384-well format, contact the ICCB-Longwood Screening Facility staff (iccb_screen”AT”hms.harvard.edu or 617-432-5815) to schedule your screen. Please contact the screening personnel well in advance of the date you would like to start your screen, as the screening schedule may be filled weeks in advance.

After establishing an assay protocol with an acceptable Z’ factor, please schedule a meeting with the data curator and data analyst to discuss data handling. This can be done by emailing david_wrobel”AT”hms.harvard.edu and jennifer_nale”AT”hms.harvard.edu.

Screening facility personnel will provide screeners with training in the use of automated liquid handlers, plate readers, and screening microscopes. Only screening facility personnel are permitted to handle library stock plates, and thus they perform all transfers from library plates into assay plates. Screening facility personnel are not responsible for conducting any other parts of a screen.

Small molecule screens: we suggest you perform an initial pilot screen of 2,000 to 3,000 wells. Additional information about libraries available for screening is available at Compound Libraries. Note that we provide an up-to-date list of recommendations (link to PDF document in first paragraph) for the order in which to screen our many small molecule libraries. If the results of your pilot screen are satisfactory, you may continue to screen additional library plates. At this point, you must submit an assay protocol for inclusion in our screening database.

When performing a screen, it is preferable to process as many plates as possible during a single visit to the screening room. We strongly suggest screening all wells in duplicate to increase the significance of data points generated. The typical daily throughput for an individual small molecule screening project is approximately 20 plates in duplicate (40 plates total, or roughly 14,000 wells) for screens involving mammalian cells, and somewhat higher for microbial screens. An average ICCB-Longwood screening project assays a total of 50,000 to 100,000 compounds for biological activity.

RNAi Screens: For human siRNA screens, screeners must first screen the pilot plate, ICCBL Pilot1 (formerly piloting utilized Mitchison Kinase1). If the results of this pilot plate are satisfactory, screening of the Dharmacon libraries can be initiated. Mouse siRNA screens begin by screening plate 50,025, the first plate in the Dharmacon Mouse1: Kinase-Phosphatase Library.

For a variety of reasons (number of replicates, time sensitivity, complexity of steps, end-point readout), the RNAi screens do not proceed at as high of a throughput as small molecule screens. It is recommended that screeners build up to screening as many as 6 library plates at a time (with this number being dependent upon the number of replicates and conditions).

ICCB-Longwood provides screening libraries and access to equipment but does not provide supplies for screeners. Screeners are responsible for purchasing and shipping all applicable supplies to ICCB-Longwood for use in their screens.

 

Data Handling

Raw screening data should be saved directly to your ICCB-Longwood server account. Detailed instructions for data formatting and deposition into our screening results database may be downloaded as a PDF from the Screening Documents page. Once data are entered into the database, instruction will be provided as to how to view your data and compare your results to those of other screens.

Your data must be entered into the database in a timely manner after completing your primary screen. Selection of compounds/siRNAs for secondary assays (“cherry picking”) is permitted only after deposition of initial data. Please see below for additional information on cherry picking.

While it is the responsibility of individual researchers to analyze their screen results, ICCB-Longwood staff can provide advice during this process. Some information may be viewed under the Data Analysis section: General Considerations and Interpretation of Hits.

 

Small Molecule Compounds for Secondary Screens

After completing a primary screen of a library, you should have many screening “positives” to follow-up in secondary screens. Subject to compound availability, we will provide you with approximately 1 µL of each selected screening positive compound (~5 µg). These are called “cherry picks”. The maximum number of cherry picks allowed is equal to 0.3% of the number of compounds initially screened. For example, if the ChemBridge DiverSet E library is the initial screen completed (16,320 compounds), a maximum of 50 cherry picks can be requested. With adequate justification, exceptions to this policy might be made; Jennifer Smith (jennifer_smith”AT”hms.harvard.edu) for approval.

Screeners with a “hit” rate higher than 0.3% will often choose their cherry picks by examining the structures of their screening positives and comparing these data with others’ screening data. This requires data entry into the Screensaver database and also time spent by the researcher mining the database. This step is usually important for the success of the project.

Requesting Small Molecule Cherry Picks:

Cherry-pick requests should be sent to David Wrobel (david_wrobel”AT”hms.harvard.edu) and are generally supplied within two weeks. Please view  Instructions for requesting small molecule cherry picks for details about submitting a cherry pick request.

Please bear in mind that cherry picks are intended to help you obtain initial confirmation of results from your primary screen, and that we do not supply material for subsequent studies. For additional material, you must order it directly from a commercial source.  Commercial vendors are often able to resupply a majority of their compounds for the first year after a library is purchased. The resupply rate varies unpredictably for older libraries. Please see the descriptions of the individual Compound Libraries for information on ordering.

For compounds no longer available from the original vendor, other supply sources may be found using public databases and other databases accessible via ICCB-Longwood. See the Interpretation of Hits section for information about these databases. Alternatively, custom synthesis can be arranged to obtain additional compound.  Rates for custom synthesis vary considerably and must be negotiated directly with suppliers.

We strongly encourage the use of analytical chemistry methods such as HPLC to confirm the identity and purity of any material reordered for secondary studies.

Strategies for testing Small Molecule Cherry Picks:

As described below, there are three options for screeners to test their cherry picks, you can download this information as a PDF, located on the Screening Documents page.

Option 1 to test cherry picks: The screener may use the HP D300 liquid dispenser to retest the 1.0 uL cherry pick volume provided by the screening facility.  The instrument uses proprietary HP T8 Dispensehead Cassettes available from Tecan.  Each cassette allows for 8 locations for solutions to be dispensed into assay plate.

HP D300

PLEASE NOTE that the only solvent supported at this time is 100% DMSO.  Aqueous solutions cannot be used with the D300 at present.

For more information on the HP D300, please visit the Tecan website here.

 


Option 2 to test cherry picks: The screener may use essentially the same assay as for their primary screen, using PocketTips ® to transfer their cherry pick compounds into their assay plates instead of pin transfer. Use of PocketTips requires lab automation and assistance from the ICCB-L staff. PocketTips allow up to 4 transfers of 100 nL from each 1 µL sample of cherry picked compound at 5 mg/mL in DMSO, but compounds at higher concentrations of DMSO (e.g., Natural Product extracts) may get fewer transfers per 1 µL.

The ICCB-L PocketTips ® protocols require that cherry pick compounds (0.6-2 µL volume) are in ABgene AB-1056 microplates, and that the plates have been spun down in a centrifuge at 1000 rpm for 1 minute immediately prior to use. The destination assay microplate should contain 30 µL of media or assay buffer. A separate PocketTip will be used for each replicate. Please inquire with screening room staff about the current price of PocketTips. The PocketTip workflow is illustrated below.

PocketTips ® Workflow

Step 1 : Source Plate: Sample aspirated and pocket in tip fills to

capacity (50 nL, 100 nL, or 250 nL).

 

Step 2 : Source Plate: Sample dispensed back to cherry pick plate and pocket remains full.

Step 3 : Wash Plate: The portion of the tip below the pocket is washed to eliminate carryover.

 

Step 4 : Destination Plate: Sample/liquid in destination (assay) plate is aspirated to pocket level in the tip, and mixed.

 

Step 5 : Destination Plate: Mixed sample is dispensed back to the destination (assay) plate.

 

Option 3 to test cherry picks: The screener may take possession of the plate of cherry pick compounds from the screening room staff and use them for follow-up assays as they see fit. Until recently this was the only option available to screeners, and many screeners elect to dilute the cherry pick compounds in DMSO or buffer to “stretch” their cherry pick compounds for more assays. Please be aware of the issues involved with retesting compounds using a higher % of DMSO. If you have any questions about this, please contact a screening room staff member.

 

Medicinal Chemistry Resources

If you are interested in medicinal chemistry support for analysis of top screening hits or for compound follow up, please contact David Scott (davida_scott”AT”dfci.harvard.edu) and The Medicinal Chemistry Core at Dana-Farber Cancer Institute.

There are also other medicinal chemistry groups in the Boston area that have worked with ICCB-Longwood screeners in the past. For more information about these potential resources, please contact Jennifer Smith (jennifer_smith’AT’hms.harvard.edu)

RNAi Reagents for Secondary Screens

After completing a primary screen of one of Dharmacon’s full genome siRNA libraries, there should be several “positives” to follow-up in secondary screens. We provide screeners with a “cherry pick” of the 4 individual duplexes targeting up to 300 genes (1200 duplexes total). Additional cherry picks can be requested at $1/duplex picked ($4/gene). Cherry picking will only be carried out for individual duplexes, not for SMARTpool reagents. Each duplex is provided at the same volume as was used when screening the SMARTpools in the primary screen. The purpose of this cherry pick is to enable screeners to perform a deconvolution screen, where the same assay is performed, but instead of using SMARTpools for knockdown, each of the 4 duplexes are tested individually.

A cherry pick may be requested at any point after screening of the Dharmacon libraries (Human 1-5, Mouse 1-5) has been initiated. Please note that it is not possible to cherry pick from the ICCBL Pilot1 plate. Cherry pick requests should be sent to David Wrobel (david_wrobel”AT”hms.harvard.edu) and are generally supplied within two weeks. Please view Instructions for requesting RNAi cherry picks (PDF) for details about submitting a cherry pick request.

If you require additional amounts of duplexes or SMARTpools, please refer to the vendor quotes on the ICCBL-L RNAi wiki for ordering information.

Other Questions

Please visit the FAQ section for answers to commonly asked screening questions, and click here to see information on how to build an academic high-throughput screening facility. If you have additional questions regarding the screening process or policies, contact the screening facility staff at iccb_screen”AT”hms.harvard.edu, Jennifer Smith (jennifer_smith”AT”hms.harvard.edu).

Please view “What Every New Screener Should Know”, suggested reading before a screener’s first visit to ICCB-Longwood.