Hi, I am using a taqman qPCR for absolute quantification of viral RNA copies in the susceptible cells at different time points after infection with virus. The way I do that is to do a standard curve with known amount of viral copies and then get the concentrations of my sample. This doesn't include any reference or house keeping gene, does anyone have any experience with this?
I have some experience here, but I would need more information to provide a useful answer. Firstly, you say you are quantifying viral RNA copies, but are these genomic RNAs, mRNAs or both? If you are studying a DNA virus (Group I, III or VI), you may be able to design primers that would differentiate between mRNA and Genomic material. In this case, you could quantify "absolutely" the levels of transcript vs genomic material for the virus. If you are studying an RNA virus, this may be possible, but will require a lot more trials.
On the other hand, perhaps you are looking for a host gene as a reference. This again is very difficult to identify as you will need to gather a lot of information regarding the degradation rates of the host vs viral nucleic acid.
In short, this is a very difficult question. Perhaps if you told us what you are trying to do it would be easier to provide suggestions
Best wishes,
Mark
Thanks Mark, here is some more explanation. Its an RNA Virus and the susceptible cells are alveolar macrophages. So I infect the cells with virus and after 3,6,9, 12,24 hours time points, extract total RNA and perform two step real time PCR using Taqman primer-probes, that are virus specific. I do absolute quantification using standard curve. In my analyses, I plot viral copies against time points. Do I need to include any controls here? to address the variables. Basically, I compare virus alone vs virus+some treatments. I aim to see the difference of viral copies that enter the cells and the rate of replication at these time points.
I see... We can make this as easy or as difficult as we want.
On the "easy" side, I would suggest the following: 1 - perform good cell counts prior to infection. 2 - either add a time point for 0 hours, or consider the 3 hour time point as your beginning point, and normalize all your samples to that time point. This will demonstrate that you have infected at the exact some MOI in each well. Then you can report your data as virus/cell at all time points.
Another, perhaps more difficult way, would be to find a host housekeeping gene THAT DOES NOT VARY over the course of infection. Then you could normalize virus to the host housekeeping gene. The difficulty, in my opinion, is that it will be very difficult to demonstrate that any host gene has NO variance during an infection. There are a number of publication showing how reference genes are picked. This involves a great deal of work. The publication below is one of my favorites. As you may want to read this and the references therein to see if this is worth your time.
Article Normalization of gene expression measurements in tumor tissu...
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