there was some problem with my influenza A probe as it was giving a peak (>35 ct) in each well, even in confirmed negative samples... i thought that probe must have degraded. So i ordered a new set of primer/probe, but the problem persists. Negative and Positive controls are working fine. Even Primer/probes for other respiratory viruses are working well...Could anyone please help me with this.....?
Hi, Hyder
Are you using serum samples or nucleic acid for influenza A quantification. If you are using serum samples, try to get the fresh blood from these patients even from the confirmed negative samples. If you are using nucleic acid for quantification of influenza A then use dilution like 1:10, 1:100 and 1:1000. I hope you have used similar concentration of nucleic acid in each sample while performing q-PCR. Sometimes companies change their flurophores. Just check what is the flurophore for ur probe set and check whether ur Machine is calibrated for this probe set or not. Try to load all your samples which had >35ct values on Agarose gel.
Best of luck..
The probe is not degraded as per your statement. I think the problem lies in the RNA extraction/elution buffer. If there would have been a problem with machine then the results would have interfered with the positive as well as negative control. Try to extract the nucleic acid with new kit/plastic ware. Place negative and positive control at different positions to ascertain the machine calibration is alright.
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