Negative Button

The negative button defines negative control wells and provides control over how negative control wells and negative control probes are used.  Negative control samples/probes may be used for detection calculations as well as potentially being subtracted from other samples/probes.

To set negative control samples, select samples on a plate with the mouse and click the negative button.
        Control Gui

The selected samples menu has three choices, "Do nothing", "Set" and "Clear".   "Set" defines the selected samples as negative controls, "Clear" removes the negative control flag from those samples.

As an indicator,  wells are marked on the plate with a solid black bar if all the experiments using that sample treat it as a negative.   The wells are marked with a small pair of bars if some experiments use it as a negative and some do not.

The checkboxes on the form define whether negative subtraction is carried out.
Negative control wells are also known as negative wells or blank wells or "water wells" (though the contents are usually not water, but assay diluent plus particles).   Negative control probes are also known as blank probes.
If water wells are defined and water well subtraction is turned on, the average across the water wells of each probe is subtracted from the corresponding probe in every well. If blank probes are defined and blank probe subtraction is turned on, the blank probe is subtracted from the other probes in each well.

The difference between water well and blank probe subtraction is most easily described by considering the data as a table with one row per sample and one column per probe.  Well subtraction means subtracting the average of the negative rows from every row.   Probe subtraction means subtracting the negative column (or average of negative columns if there are more than one) from every column.   See data processing.

It is also possible to set negative control samples on a per-experiment level, by toggling the negative flag on the sample table for a given experiment, and similarly for blank probes.

Subtracting either probe values or well values from other values may lead to negative signal for some targets in some wells.  If it is desired to truncate such values to zero (or any small positive number) it can be entered in the box marked "Zero level".   A small positive number may be preferred for log plots.

Negative control probes are usually recognized by names such as "Neg. Ctl." and automatically marked as such; if a probe is not so marked and is desired to be treated as a negative control it can be checked on the probe table.

When using normalization, a negative sample, that is not marked as such, is undesirable in an experiment, since normalization will bring it up to the level of the other samples and make it look as if it has high levels of expression.

The use of negatives, blanks and normalization is described in the data processing document file.

See Also

Data processing
Minimum detectable dose

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