I am working on a multiplex qPCR to detect simultaneously a viral RNA target and an exogenous RNA internal control using hydrolysis probes. As you can see from the picture, this internal control was synthesized using the viral RNA sequence as template and adding a random sequence in the middle, where the specific probe to this internal control binds. As shown in the picture, the probe used to detect the viral RNA should only bind to the target viral sequence. However, when testing a virus (-) / internal control (+) sample, a non specific signal is observed with this probe. How can I overcome this issue? Where is it best to "cut" my probe? 5´or 3´? I take any advice, thank you very much
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