Recently I did qPCR using mice xenograft tumor samples. I got very good amplification of internal loading control GAPDH but not my target gene using human Taqman probes. (although I am not sure at this stage whether its solely because of primer specificity or some other factors involved). Since xenografts contain a mix of human and mouse tissues, I was wondering which species-specific primer I should use for my qPCR assays? and what is the best strategy to get good qPCR data with such samples? Any suggestions would be appreciated.
In case your PCR is qualitative: If the gene you are trying to quantify is highly conserved (high horology between human and mouse sequence), you may use a degenerate primer pair (a mixture of primers having the human gene sequence and the mouse gene sequence.
If your PCR is Quantitative: more information is needed about what you are trying to do? You may need to do PCRs for both and then you can determine what proportion of the tumor has come from the human or mouse. If you are interested in the human gene alone, then you need to detect the gene you are interested in.
Regarding the protocol, it depends on the kit you are using. I recommend following the protocol provided by the manufacturer for a start, particularly if you are using a two-steps amplification reaction.
Hello Sajid,
First of all you should check the specificity of the primers. If they are specific then check the melting temperature or try a gradient first to see if your primer is able to amplify the gene of interest.
There no special way to address it. Follow the basic steps, i.e primer specificity, its melting temperature, cDNA concentration and use a positive controls. For positive control you can use cDNA from human cell line or a cell line derived from same PDX line using your primer of interest.
If your primer and probe sets work in positive control, then they should work in your PDX samples as well. If they are not, then increase the cDNA concentration or prepare fresh samples and try. Your target gene could be low abundance. If that is the case and you have limited sample, try a re-PCR.
Best
Ravi
Sir, try to set best temperatures. Check all in a range for optimisation of primer. For annealin. Am sure it will work. Mouse origi preferred as samples are from mice. Thanks
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