How to handle no Passive reference case in qpcr experiment

Question

[venkatreddiee]

In case of no passive reference dye(ROX) in a qpcr experiment, how instrument handles background correction? We are using ThermoFisher's QuantStudio 3 and 5 instruments.

If instrument does not do any background correction in this case, how the data should be processed for further analysis?

Normalizing the fluorescence intensity is accomplished dividing the emission intensity of the reporter dye by the emission intensity of a reference dye. Without background correction using ROX fluorescence, data looks off the charts.

Answer 1

[theobroma22]

Sure, you can look into qPCR and the Ct methods. QPCR uses housekeeping genes to normalize gene expression values - depending on your experiment such as time course or control vs. treatment you have the option to look at 2^-ddCt or delta Ct normalization. Ct stands for count threshold.

Answer 2

[theobroma22]

You cannot normalize the data without background correction. So, without ROX you do not have a reference to normalize the reporters. Unless your reporter set included some housekeeping genes, currently normalization is not achievable. You could use the housekeeping genes expression, which should be constitutive, as your reference to normalize the expressions of the reporters.

Answer 3

[venkatreddiee]

@theobroma22, Thanks for your reply. Since the house keeping genes have some expression of their own we may not be using whole intensity as equivalent to background. Is there a way you can think of using/implementing these house keeping genes fluorescence to do background correction or normalization? Please let me know if an existing or an experimented approach in this case.

Answer 4

[venkatreddiee]

Thanks so much. That's exactly what I am looking for, ∆∆Ct to normalize the data. This seems more appropriate for my condition. I will discuss with my team and hopefully my issue solved for now. Really appreciate your help @theobroma22.