Reference Gene Normalization

Use this option to specify housekeeping genes or stable genes with low coefficient of variation to compute the normalization factors.

Select Housekeeping Genes to manually select a list of target sequences that are consistent in their expression levels and are either recognized by the CodeSet (marked as Housekeeping, for example), or according to the user’s prior knowledge. 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 you specify, the more accurate the normalization.

Select Stable Genes to select a list of less variable genes. To identify these genes, this process automatically calculates both the average expression and the coefficient of variation (CV) of all the genes. It then selects genes with average expression >50th percentile and CV<50th percentile as candidate list. Finally, it ranks them by CV and picks 10 or fewer genes with the lowest CV values to use as reference genes.

Select None to use no reference genes for normalization.