Normalize to baseline

If your experimental design includes a sample or a group of samples serving as a baseline control, you can normalize the experimental samples by subtracting or dividing by the baseline sample(s) using the Normalize to baseline task in menu. For example, in PCR experiments, the delta Ct values of control samples are subtracted from the delta Ct values of experimental samples to obtain delta-delta Ct values for the experimental samples.

The Normalize to baseline task is available in the Normalization and Scaling section of the context-sensitive menu upon selection of any count matrix data node.

There are three options to choose the baseline samples:

• use all samples

• use a group

• use matched pairs

Use all samples to create baseline

To normalize data to all the samples, choose to calculate the baseline using the mean or median of all samples for each feature, and choose to subtract baseline or ratio to baseline for the normalization method and click Finish.

Use a group to create baseline

When there is a subset of samples that serve as the baseline in the experiment, select use group for Choose baseline samples. The specific group should be specified using sample attributes.

Choose use group, select the attribute containing the baseline group information, e.g. Phenotype in this example, the samples with the group Normal for the Phenotype attribute used as the baseline. The normal samples can be filtered out after normalization by selecting the Remove baseline samples after normalization check box if you don't want to include normal samples in the downstream analysis.

Use matched pairs

When using matched pairs, one sample from each pair serves as the control. An attribute specifying the pairs must be selected in addition to an attribute designating which sample in each pair is the baseline sample.

After normalization, all values for the control sample will be either 0 or 1 depending on the normalization method chosen, Subtract baseline or Ratio to baseline respectively, so we recommend removing baseline samples when using matched pairs.