Hi , I want to know that if I hypothesize that Gene A promoting chemoresistance in PDAC but what to do if I do not get any positive staining in my sample? What measure will I have to take which will guarantee me to get atleast something to work on?
First of all, as you said it is your "hypothesis" which itself means it is subject to experimentation and if not found to be yielding results, then the idea is simply to be discarded. You cannot force your hypothesis. However before discarding the idea altogether, you should explore from all angles. Since I do not know what experiments you have done to check this, I would suggest some which might help you.
If you still do not see any expression in all the above cases of your gene A, then you may try the following experiments:
I do not exactly know your setup, but I am sure any of these would help you. If not, you may share your experimental plan, so that we could think of other ways. All the best!
Thanks Dr.Anupa, i haven't yet started the project.I only made a research proposal. Yes my experiments along that line. I will extract patient's tissue followed by IHC and will do some expression analysis at RNA and protein level. Silencing and overexpression experiments are also a part of my project.
G. Anupa brilliant suggestions. Further, I would like to add some points and considerations for Tooba Jawwad :
1. Is it worth doing (I mean is that gene that important to you?) all the experimentation just to determine that that GOI is involved in chemoresistance.
2. If not much, you can simply switch to some other gene or molecular mechanisms.
3. If yes or in case you have committed its exploration), I will recommend suggestions from Anupa.
4. Another thing is you better troubleshoot and make sure that whether your sample surely does not have its staining/ protein level, and if possible mRNA level. As there might be cases that mRNA is present and differentially expressed, but there is no translation and hence no protein.
5. Another interesting possible thing. We recently reported that DNA Ligase 4 is downregulated in colon cancer, while a previous report is already available where its overexpression in some specific cells promotes tumor, so cell specific expression is an important consideration. Thats why single cell RNA-seq is being preferred and providing novel insights regarding role of genes which were earlier reported to have no change in expression , but are actually important regulators in cancer.
Hope this helps.
Further, I am getting similar results in my project. So I can understand. All the best.
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