The Reference Gene Normalization process is performed in order to adjust counts of all probes relative to a probe (or set of probes) that are not expected to vary between samples or replicates.
This process assumes that some of the target sequences recognized by the CodeSet are consistent in their expression levels. The choice of reference genes to include in the CodeSet is therefore a crucial part of experimental design and Nanostring recommends selecting at least three reference genes. However, you should keep in mind that the more reference genes that are specified, the more accurate the normalization.
One Input SAS Data Set is required by this process: The edf_wide.sas7bdat data set serves as an example, and is shown below. This data set was generated by running the Nanostring Input Engine process using the edf.sas7bdat experimental design file and the raw .RCC files are located in the Sample Data\Nanostring directory that is included with JMP Genomics.This is a wide data set, with 6 rows listing individual samples and 66 columns containing count data.
The output data sets generated by this process are listed in a Results window. Refer to the Reference (Housekeeping) Gene Normalization output documentation for detailed descriptions.