Author Topic: help please I am lost  (Read 2755 times)

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Offline Clear

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help please I am lost
« on: December 11, 2010, 03:44:07 AM »
Hi all
since i started my PhD I have done lots of experiments using IHC fluorescence to identify different cellular phenotype in neural stem cell line every experiment have been replicated three biological replicants. to confirm my result with other tool I asked i collegue to help me in Qpcr and he kindly run this for me which gave me odd result. in addition to completely contradict what i have found in IHC it suggest that the cells express certain gene I would not expect the cell to express it based on the review of  literature. the qpcr is one biological replicant so far with three technical replicants. my college was complaining fron the start that he have used three house keeping genes which were not helping each other.  I am not expert in Qpcr and really worried regarding the fact that the qpcr data contradict some of the key result in my IHC. any idea what would be the reason behind that.I really would appreaciate your precious time and advise. since have this result I really can not sleep
thanks in advance

help please I am lost
« on: December 11, 2010, 03:44:07 AM »

Offline excalibur

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Re: help please I am lost
« Reply #1 on: December 14, 2010, 10:14:38 AM »
Are you running known controls from tissues other than your experimental tissue? Always run controls to optimize the antibody and confirm that it works.

Have you done chromagenic staining with AEC or DAB to compare and contrast with the fluoro?
Paula K. Pierce, HTL(ASCP)HT
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Offline Cardio

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Re: help please I am lost
« Reply #2 on: December 14, 2010, 04:04:31 PM »
So I am having a hard time understanding your problem.

Is it that you see no protein via IF but you do see RNA? If thats the case then it could boil down to some post transcriptional modification that you are seeing.

Excalibur is right. You need to make sure you have proper controls before you rule out a negative staining. You can also do western as well to see if you have protein.

Offline Clear

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Re: help please I am lost
« Reply #3 on: December 15, 2010, 01:13:26 PM »
Hello
I am sorry if I confused you, It is the other way around I have protein expression by IF IHC and found the number of certain type of cell in my treated culture more than control(this is have been done three biological replicants) when I tried to confirm that by Qpcr (one biological replicant so far) the result of the qpcr did not support my IHC results with the control showing more gene expression than the treated. I am not expert in Qpcr but the colleague who run the Qpcr for me was cmplaing from the house keeping genes that they are not support each other.
Many thanks again, looking forwards to hearing from you

Offline SarahG

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Re: help please I am lost
« Reply #4 on: July 15, 2011, 12:26:27 PM »
Hi Clear,

It has been awhile since you originally posted your question so hopefully you have already found a solution. In case someone else has a similar problem, I thought I would post a link to a paper that talks about the influence that reference/housekeeping gene choice can have on qPCR results and some approaches that can be taken to help identify suitable references genes for each experiment.

http://www.biomedcentral.com/1471-2407/9/49

If your colleague is concerned that the housekeeping genes used for qPCR are not showing stable expression across the different treatments or conditions you are examining, it is possible that one or more of those housekeeping genes may not be ideal for this particular experiment.
Sarah Gonsalves, H.BSc.
Ph.D. Candidate, University of Toronto
Intern, 1DegreeBio
http://1degreebio.org

Re: help please I am lost
« Reply #4 on: July 15, 2011, 12:26:27 PM »