I've been working for a long time with this probe set routinely and results in general are very good since the protocol has already been standadized. Nevertheless I have been getting this kind of results lately and im not quite sure what could be happening.
Is this a reagent degradation case? could it be the internal control probes or dyes from this reagent batch wich are causing trouble? I also see that there are some RFU values that go bellow the baseline background for this probe.
what do you think about it?
Your answers are always valuable and Im very gratefull for your help :)
Hi Christian,
It seems your amplification is starting after 40 cycles, it should reach saturation at this stage. Are you sure that enough concentration of cDNA or primer is used? I would recommend you to perform a normal PCR with the cDNA and the primers for your gene of interest as well as housekeeping genes, before going to qPCR.
Best of luck!
I'm guessing your reagents have degraded. You are getting very little amplification from any of the samples.
Get a fresh batch of reagents & try again.
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