I have quantified plasma protein (ELISA) and mRNA (whole blood; RT-PCR) for a candidate molecule. Independently, a comparison of plasma levels between 2 groups shows a significant difference; whereas, comparison of dCt is not statistically significant. Also, dCt vs conc ng/ul shows a negative correlation.
1. Should I compare dCt between the two groups or rely on fold-change to assess whether my molecule is expressed in the case group vs the control group?
2. How do I justify the negative correlation obtained while comparing plasma protein levels vs whole blood mRNA levels in the same set of patients?
You have two sets of numbers. You can compare within each set (group 1 vs group 2) and also compare between sets (biomarker 1 vs biomarker 2).
It seems like you've done this, so...I don't see the problem.
Provided your within-set measurements are valid (for protein: equal vols of plasma between groups, and/or normalised to a reference biomarker; for mRNA: correctly normalised to reference genes) then all your comparisons are fine.
Your data is your data: don't try to force a model onto it, let the data inform your model. If mRNA doesn't correlate with the protein biomarker, then...maybe that biomarker is being made elsewhere and released into the circulation. There are plenty of biomarkers like this: in muscular dystrophy, for example, you see elevated blood creatine kinase, cardiac troponin, miR-206, but none of these are _made_ in the blood, they're all things that leak out of damaged muscle.
Important: correlate dCt values to log conc.
Also be aware about how you calculated dCt. If you subtract the REF Ct from the GOI Ct (some people strangely do this), then higher dCt values indicate lower GOI expression (assuming REF expression is constant)!
source of the plasma protein might not be from the cells in your whole blood sample so the levels may not correlate. if you know that this protein is majority generated from the cells in the whole blood then I agree with Jochen Wilhelm . keep in might the directionality of your calculations and the fact that protein levels do not always correlate with mRNA levels anyway.
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