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How is the nCounter Binding Density QC metric interpreted?

Binding density is the number of fluorescent spots per square micron that are bound on the imaging surface for a sample in the nCounter cartridge. The upper threshold for flagging samples as having high binding density tends to be conservative and is typically not interpreted as a strict pass/fail metric. Binding density can be affected by several factors:
  • RNA input mass (ng)
    • The relationship between binding density and RNA input (ng) is generally linear until the point of imaging surface saturation, all else being equal. Loading higher amounts of RNA into the assay tends to result in higher binding density. Conversely, loading lower amounts of RNA (ng) tends to result in a lower binding density. Factors that may impact the amount of RNA loaded into an assay include inaccuracy in quantification or degradation/fragmentation. If using cell lysates as input into the assay, then inaccuracy in cell counting or stimulation/repression of transcription (that changes the mass of RNA present on a per cell basis) may also impact binding density across samples in an experiment.
  • Number of targets
    • In general, as the number of probes/target genes in an assay increase, the binding density also increases. As the number of probes/target genes decrease, then binding density tends to decrease.
  • Expression level of the targets
    • Quantification of highly expressed genes will tend to lead to high binding density, whereas quantification of low expressors will tend to lead to low binding density.

If binding density is high, then the barcodes bound on the imaging surface can start to lay close to each other and even overlap. Checking the Positive Control Linearity and Limit of Detection QC metrics would be a prudent next step to determine if data linearity or detection of low expressers was impacted. In extreme cases of high density, the imaging surface becomes saturated with barcodes, and the imaging begins to fail. For help with interpretation of data flagged for high binding density, please contact NanoString Support () or your local NanoString representative.