Verification of protein level change by RT-PCR - the results are just not compatible (Sep/11/2006 )

I found several protein changed by 2D by comparing control and Durg treated group of gels.
Foutunatelu, I found several of them, I first verify them by 1D western blot, however, it seems that the 1D bands shows opposite direction of protein level change.
I use Real time PCR to verify the same protein on the gene expression level, however, there are also sevel gene changed in oposite direction.
Does anyone encounter such problem?
How can I interpret my result?

Thanks a lot.

I believe the result are true result, but the way to interpret is head cracking.

---

Keep in mind that mRNA levels and protein levels are different!
mRNA does not mean that this becomes a protein, it's supposed to be, but it can be interfered with a lot of other things. And also proteins can be degraded by proteases.

---

Yes, I also agree that, is there any paper have mentioned this point and give a conclusive comment on this?
For RT-PCR, we use the cDNA sequence, which is translate from the protein sequence, they suppose to be transcript and be a protein, what process could interfere this procedure?

BTW, why my 2D result can not be verified by 1D westerblot of the specific protein, could be that the antibody recognize several isoform of this protein?

---

I cannot answer your question about the gels.
But when mRNA is present, it's translated into a protein. There still several modifications take place and the protein needs to be folded. If there's a problem, the (not-yet existing) protein is broken down. It can also be broken down by proteases. So the mRNA could be higher, while the protein itselve is degraded.

---

In my study, I test the HSP27 level change after treat with a drug, the protein level is up regulated by WB, but the RT-PCR result show that it is 0.22 fold down regulated. The result was repeated 3 times, each time is same outcome.

I belive in them all, but I must explain why such phenomena happen?
Shall I buy new antibody and test the protein by WB?

The protease theory can not work in this situation.
Any other explanation?

Thanks a lot.

---

Anybody know any paper or journals claim that they found out the protein level change and gene expression level change are different?

Due to my own experience, I found people only report the target gene that change in same direction in protein expression level and gene expression level.

---

This is the other way around indeed.
I don't know what the half-life of the protein is. Changes in mRNA levels can be seen very fast after drug treathment, maybe there was still HSP27 present. Or did the protein itself was upregulated on WB? I also only know that either both were upregulated or that the mRNA was upregulated but not the protein, this I have never seen.
And are you sure that the antibody is specific, that it does not bind to other proteins out the HSP family?

---

Maybe I should test the protein and mRNA level change by time course, so know it clear.

I am also worried that the antibody may be wrong, so I think I should buy a new one and test.

The protein was clearly upregulated aftern treatment shown in WB, but the RT-PCR fold change in treated sample is 0.22, down regulated.

I do feel it is very difficult to interpret this phenomena.

COuld that due to phophorylation of HSP27?

---

I would definitely do a time course. It is very likely that your mRNA increases at a different time then your protein (we'd expect mRNA increases prior to protein increase).

---