Why am I seeing the QC warning 'Low percentage of housekeeping genes over 50'

Your NanoString panel/codeset will include multiple housekeeping (HK) genes to be used for normalizing your samples. The exact number of HK genes will vary depending on the panel. 50 counts is an approximate number counts that is used as a threshold for HK genes. The background noise threshold is calculated from counts of negative controls. In most cases, counts below 50 are considered too close to background noise for genes to be used for normalization.

This ROSALIND QC flag 'Low percentage of housekeeping genes over 50' means that many of the HK genes in the panel resulted in counts too low to be used for normalizing your samples. However, they were still able to be normalized using other HK genes available. In ROSALIND, you can check which specific HK genes (and how many of them) were used for your analysis by downloading the .CSV file from the QC plot 'Variance of Mean Notes'. For visuals on how to download this list, please see here.