Protein expression but no mRNA

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    • #16101
      evamik
      Participant

      I am looking into the expression of a certain gene that is clearly overexpressed both at the gene (RT-PCR) and protein (Western blot) levels in some malignancies. However, when I test healthy controls, they are clearly positive in Western blot but completely negative in RT-PCR. I have used several different primer combinations so the reason for the negative RT-PCR is not e.g. point mutations. So, does anyone have a suggestion to how a protein may be detected while there seems to be no mRNA.

    • #109689
      JackBean
      Participant

      non-specific antibodies?

    • #109774
      evamik
      Participant

      Good point, but the detected protein is a single band of the expected size so I can not really believe that it is unspecific binding.

    • #109776
      JackBean
      Participant

      and did you tried your primers with some positive control?

    • #109784
      jonmoulton
      Participant

      If you have a protein with slow turnover and its expression is inhibited by the activity of the protein (e.g. end-product inhibition) then the protein level could be high but the mRNA very low. If your healthy controls are not growing (e.g. confluent cells in culture) you might try reassessing the mRNA when the cells are growing more rapidly (split the cultures and assay before they are confluent). Active growth should cause the cells to make more of the mRNA & new protein as the protein will be needed to keep the cytosolic concentration near constant.

    • #109803
      evamik
      Participant

      Absolutely! And the patient sampes tested are clearly positive.

    • #109804
      evamik
      Participant

      Good point!! But the cells I work with are primary cells (lymphocytes isolated directly from fresh blood). The leukemia I work on is characterized by a SLOW progression only partly due to proliferation and even more due to inhibited apoptosis.

      Is endocytosis a completely unthinkable (and maybe stupid) suggestion?

    • #109805
      jonmoulton
      Participant

      Are you suggesting that the protein is being endocytosed rather than expressed in the cultured primary cells? I doubt that would be the case because on fusion of the endosome with a lysosome the proteins in the vesicle are usually degraded by the lysosomal enzymes. For the protein to enter using endosomes, it would have to either resist proteolytic degradation or prevent fusion of the endosome and lysosome. Further, the protein would have to escape from the endosome if it is to function in the cytosol. It may not be impossible for an intact protein to both survive and escape from the endocytotic pathway, but it is not common.

    • #109806
      JackBean
      Participant

      Furthermore, if you’re talking about leukocytes, the protein’d have to be present in blood serum.

    • #111487
      evamik
      Participant

      Sorry for not responding sooner.

      The protein is indeed present in serum. A strong band is detected both in patients and in healthy controls. The molecular weight in serum is the same as seen in cell lysates. Maybe, the protein is simply bound to cell surface receptors?!? But when I do fractionation studies, the protein is detected in the cytosollic fraction. More ideas?

    • #111495
      jonmoulton
      Participant

      I suppose isotopic tracing to detect the protein origin is out of the question…

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