Put simply, you can't. Some people may use the maximum cycle number to replace an undetected value, but this is bad science. You don't know if your target was undetected because it was not expressed or if your reaction didn't work.

I would simply run your qPCR products on a gel and show the absence/presence of a band.

This is a pretty common question. Here a couple similar posts that might help:

https://www.researchgate.net/post/Is-there-a-way-to-calculate-ddCts-fold-changes-if-your-gene-of-interest-is-undetectable-in-unstimulated-but-then-detectable-after-stimulation

https://www.researchgate.net/post/How-to-deal-with-non-detects-in-CT-data-when-performing-Delta-Delta-CT-Method

Thank you so much for your response John Hardy Lockhart !

Sorry to bother you with more questions. I wonder if there is some other method to present my data? The scenario here is that I am looking if certain cells can be infected with a certain virus. My reference sample is non-infected cells, reasonably, there is no expression of viral genes. The reference gene is expressed in this non-infected sample (meaning the cDNA is fine), while all the viral target genes are beautifully expressed in virus donor cells (meaning the primers for viral genes are fine). So can I tell that the reaction, after all, worked and the viral targets are undetectable in the non-infected sample because there is no expression whatsoever?

Thanks a million!

Hi,

I agree with suggestions of prof. John above, that it is not appropriate to use undetermined state of reference gene for any calculations. For comparable quality of isolated RNA or cDNA prep. from non-infected or infected samples, you may be able to rely on reference genes specific to the host RNA (and not viral genes). As you already mentioned the viral genes will be specifically present in the infected sample. Common host genes will indicate that the RNA quality is fine in both samples.

And you may also use 'Absolute quantification' qRT-PCR method for estimation of viral transcript in both samples using a standard curve of the 'viral gene' itself.

I think Ajay Saini has the right idea. If you're looking to compare infectivity among different cell lines, you'll need to use absolute quantification qRT-PCR. If you're just trying to show if a cell line can or cannot be infected, showing the PCR bands should be sufficient. Either way, including a known infectable cell line as a positive control will assuage any concerns that the lack of a band is due to PCR failure.

Hey Ajay Saini, thank you for taking your time and answering my question. I think you misunderstood me - in the undetermined state is my reference sample (calibrator sample) and not the reference gene. My reference gene is in fact expressed very distinctly in this non-infected calibrator sample. The reference gene that I used is acidic ribosomal protein 36B4 from the human host and not a viral gene.

I was just wondering if I could still use the data from the comparative qPCR study that I already completed or do I need to rerun a standard curve assay?

Thanks again,

Best

John Hardy Lockhart, I am afraid I don't have the possibility to rerun an agarose gel. Could the bands be replaced with melt curve data to show the presence and absence of an amplicon? My apologies if this sounds nonsense, I am new in qPCR and trying to figure out things.

Could I use the delta Ct method or my results are of no use? Do I need to repeat with a standard curve method?

Thanks a mill!

While melt curves can provide some of the same information an agarose gel, I don't recall ever seeing them published as a stand-in for bands on a gel. That being said, I would hold on to those data in case you need to provide additional support for your conclusions.

You can potentially show the ∆Ct of your viral proteins, but you're still going to have the issue of an undetermined value in the uninfected samples. If you take at this other post (https://www.researchgate.net/post/Is-there-a-way-to-calculate-ddCts-fold-changes-if-your-gene-of-interest-is-undetectable-in-unstimulated-but-then-detectable-after-stimulation) you'll see recommendations about how to report this sort of data. It's not as "convincing" as showing actual bands, but qPCR results generally have to be accepted in good-faith anyways.

Hi

I agree with the points above, and clarification by Lilit.

The melt-profiles can be used to ascertain the presence/absence of an amplicon, however, as pointed by John here are few papers in the literature on use of amplicons. However, you need to first show association of a melt-profile with the band/fragment of interest. Then you can use it, and for a single specific amplicon it is actually useful and saves time