real time data and elisa data connection

[anu04]

i am working in area of wound healing. i am estimating various cytokines using real time pcr , elisa and western blotting.how can i correlate real time data and elisa data?

[mchlbrmn]

Maybe you could use one sample to normalize to(?). For example, the resting tissue can be normalized to "1" in the various methods, and the treated (injured?) samples are expressed as ratios of this. Would that make sense? For the qPCR the data would first be normalized to the housekeeping gene to control for sample loading, and then normalize to the control tissue sample. I suppose that might also be done for the ELISA, but I've not experienced there.

[relaxin]

RT-qPCR measures cytokline mRNA levels and ELISA and Western measure their protein levels. You may or may not see a parallel rise and fall of their mRNA and protein levels, depending on their stability. As long as you are using the same control and comparing the basal level with those of treatment, you will get a nice story.

[anu04]

according to my real time data tnf alpha has increased expression in treated groups than control but according to elisa tnf level is less compared to control.,,.what could be the reason?

[mchlbrmn]

I don't know.
Are you sure your "housekeeping" gene used to normalize the qPCR is not regulated in this experiment?
The PCR is for certain primer sequences located in one part of the gene. One possibility is that there are alternately spliced transcriopts of the gene produced. there could be more total transcript, but less protein producing transcript produced in treated groupd, or less protein that binds the antibody used in the ELISA. For example, an alternate splice may have a different promoter giving it different tissue specific expression, or an alternate exon may be omitted from one transcript that is the location a monoclonal antibody binds to.
There could also be regulation at the translation level, by non coding RNA for example, or by an RNA binding protein. There could be factors affecting RNA stability.
The primers could PCR mRNA, but they may also PCR non coding RNA. For example an antisense RNA upregulated in treated tissue could increase RNA /PCR product level, but might inhibit protein expression. (Don't ask me how common that is).

[anu04]

thank u for your valuable comments..let me try once again............

[relaxin]

according to my real time data tnf alpha has increased expression in treated groups than control but according to elisa tnf level is less compared to control.,,.what could be the reason?

TNF protein may be less stable than mRNA.

How many fold increase in mRNA level? How many fold decrease in protein level? If both are less than 2-fold, I do not think the difference will be significant.

[mchlbrmn]

Good points.