I would like to use an exogenous internal positive control in my qPCR reactions (probe-based with gDNA). The IPC protocol states a 60C anneal/extend step, but my target assays require this step to be 63.3C.
I tried running the IPC at 63.3C (singleplex, not with my assays) to see how it performs at this higher temp. I had previously tried running one of my target assays (FAM) with the IPC (VIC) at 60C using the manufacturer's protocol.
The average RFU at 63.3C (IPC alone) was 765, and the average RFU at 60C (IPC + FAM assay) was 6700 and 9400 (tried it twice on separate days).
The call type was as expected (positive call for all reactions except for those with the IPC blocker), but the RFU values are very different.
Does it matter that the RFU values are so different? Is this difference because the reactions were run in singleplex vs duplex (lower fluorescent signal?) or because the temperature is higher? I just want to confirm the IPC's performance and determine whether or not I can reliably include this control in my reactions.
Product used: TaqMan™ Exogenous Internal Positive Control Reagents (thermofisher.com)
Purpose: to test for inhibition (sample is avian feces).
I think that lower fluorescent signal is the result of not having optimal annealing temperature for IPC probe, not result of duplex reaction. We had a similar problem. We have IPC from other producer and it runs on 60C but not on 65C that was needed for my qPCR. We could not optimize the reaction so we changed IPC (used constructed one).
With this drop in fluorescent signal, though, do you think I could reliably use the IPC at 63.3C within each sample reaction? Or is the performance affected enough that I should not attempt to include this IPC in my reactions under these non-optimal conditions?
Note: the neg/pos calls were as expected (i.e., NTC, NAC, spiked fecal matrix).
I think it depends from what you are doing. We were unsure if the later Ct values of the IPC are results of inhibition of PCR reaction or from the sub-optimal PCR conditions. But, in our case difference in annealing temperature was 5C. Maybe you can still go with it, just need to repeat several times to see if the results are repeatable.
If you are doing official diagnostic testings I would recommend to go with it.
If you are doing official diagnostic testings I would NOT recommend to go with it.
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