Hello,
I am an graduate student from Korea republic.
Before asking a question,
I remeber those who answered my previous questions and
I want to appreciate them. All answers are great help for me!
Here is my question : I am working with huvec and
I am always cautious about other cells mixed with huvec.
So I am planning to conduct real-time pcr with primers of
huvec markers(vegfr1 and vegfr3) and gapdh(control) to see whether
cells I have worked with are huvec.
I wonder that mRNA of vegfr1 and vegfr3 can function as huvec markers.
I think that it is the best to do with protein level but
I do not have antibodies to do the work(I am yet an young researcher)
Thank you!
Always correlate RNA data with protein. In our lab, we can often get excited about 2 or even 10 fold increases in messages of gene of interest, but by western blot, or by FACS we could see (depending on the gene) either no increased protein, no protein whatsoever, or such a subtle difference that you could not get a p-value, while the real-time PCR would give us p-values.
If you don't see the RNA, you may or may not have protein do to mRNA turnover rates. And increased RNA isn't always indicative of increased protein expression.
I agree, RNA is probably not the right choice. I'd use a genetic marker to track them, like microsatellites. If it's a short term thing, you could also stain the HUVECs, but not whatever you're mixing them with with Calcein AM. Lasts about 2 days. Or if whatever you're mixing them with is clonal, consider inserting a GFP genetically.
If I get it right you think that your huvec cells are somehow contaminated or mixed with other cells. So if it is possible I would try to get real and clean huvec cells from another lab or a company. I think this saves time and maybe money. What is your plan if you find out that you have mixed a cell population?
I also suggest to get confirmed clean cell lines from appropriate sources, rather than figuring out a suspected contamination.
If this is not an option, real time PCR is ok to determine expression biomarkers. you do not need protein assays.
I am not an expert in HUVEC specific gene expression. however, if you determine HUVEC specific markers, how do you figure out if you do have contamination in your cell line? The HUVEC markers will always be there. you need to know the identity of the suspected contaminating cell line, and determine specific genes they express, in order to confirm contamination, I suppose?
Maybe you could consider karyotyping?
that will all take time, so at the end, try to find an uncontaminated cell line.
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