Tech Notes

Q. What normalizations do I need to apply to Real-time PCR (RT-PCR) data before I compare them to my microarray data?

A. It is very important to check normalization settings after importing data especially since the defaults applied are more suitable for microarray data than for RT-PCR data.

If you are importing delta-delta-CT values, you first need to apply the RT-PCR data transformation. If you are measuring over numerous conditions (> 2), then you may also want to follow this transformation with the 'Per-Gene' normalization which centers the profiles to a baseline at y=1.0

If you are importing measurements already converted to values correlated with RNA levels, then only a 'Per-Gene'-normalization is recommended.

Watch out for the data transformation 'Transform values <0.01 to 0.01' that appears among the default normalizations. The cut-off should either be lowered significantly to something more reasonable for RT-PCR data, or more likely, it should be removed altogether.