Many of the dose-response and quality control tools in biochemical and phenotypic HTS screens rely on controls, so the first thing to do, is to define how your controls are laid out on your plates for a given protocol.
You can define positive, negative and reference controls in CDD:
- Positive Control: Shows strong activity (inhibition or activation). This control is used as a high control when normalizing raw data to calculate % inhibition.
- Negative Control: Shows no activity. Depending on your assay, , this can be e.g. a "blank, solvent, or "cells only" control, etc. This control is used as a low control when normalizing raw data to calculate % inhibition.
Based on those definitions, negative controls might have higher values than positive controls.
- Reference molecule: Also referred to as a "benchmark" compound, is typically a known molecule that's been tested in a particular protocol many times and demonstrates reproducible activity. Therefore it can be used as a protocol quality metric. This control is not used to normalize raw data.
If you plan to use benchmark compounds to normalize raw data, you must set it as a positive control instead.
The reference molecule curve will be be overlaid on each sample plot of the run.
Define Control Plate Layout
CDD supports 96-well, 384-well, and 1536-well plates or a mix of them. If all your plates (or most) follow the same layout, the first thing to do is specify a protocol-wide default control layout for your plate size. This play layout will apply to all plates across all runs of your protocol, but don't worry, you can over-ride this default with specific plate layouts.
Scroll down to the bottom of the Protocol details tab and you will see three collapsed sections: Protocol Default 96-well Control Layout, Protocol Default 384-well Control Layout, and Protocol Default 1536-well Control Layout. Expand the one you want to modify, click the edit link and follow the simple instructions.
1 click for positive control
2 clicks for negative control
3 clicks for reference substance
4 clicks back to empty
Now that you have a default layout, you can define control layouts for any specific plates that are different. These might include the final incomplete plates of a library or special control plates. In any case, click the “Add a plate-specific control layout” and follow the instructions. If you have not yet imported your plate maps, you can come back after you have done so.
If your plate control layouts are different for each run, you can define them on the Run details tab of each run page.
Control Layout Tips
- Control layouts can be added after importing data and can be changed whenever you want: all calculations are dynamically updated for the relevant plates.
- Control layouts are always displayed on heat maps, which provides a convenient way to double-check that the correct layout is being applied.