We have transfected HEK293 Cells with a viral gene. I have extracted genomic DNA from the mammalian cells. How can I determine the viral gene copy number using qPCR? Thanks in advance for the information.
HI...
To determine the viral gene copy number in genomic DNA extracted from transfected cells using qPCR, you can follow these steps:
Design specific primers for the viral gene of interest. The primers should be specific to the viral gene and should amplify a specific fragment of the gene.
Set up a qPCR reaction using the genomic DNA as the template, the viral gene primers, and a suitable qPCR mix.
Run the qPCR reaction according to the manufacturer's instructions.
Determine the viral gene copy number by comparing the qPCR results to a standard curve. To do this, you will need to prepare a series of known concentrations of the viral gene and run qPCR reactions for each concentration. The known concentrations should cover the range of expected copy numbers for the viral gene in the genomic DNA.
Plot the qPCR results for the known concentrations on a graph, with the concentration on the x-axis and the qPCR signal on the y-axis.
Draw a standard curve by connecting the points on the graph with a line.
Determine the viral gene copy number for the unknown samples by finding the concentration on the x-axis that corresponds to the same y-axis value as the unknown sample. This will give you the viral gene copy number for the unknown sample.
I hope this information is helpful.
Regards
Hi Dr. Rathnayake,
Hopefully, the brilliantly detailed explanation of Dr. Mohammad Alzeyadi should've made the procedure crystal clear to you.
I thought sharing what I am doing now with you might be handy as well.
So I am working on some high-risk HPV types. I am using NIBSC's standard DNAs (which have known copy numbers per microliter) to generate a standard curve based on which the viral copy numbers in my patient samples can be estimated.
Hope that helps.
Best of luck for the rest of your scientific journey,
Negar
Thank you Negar Fakhari for the information. Good luck with your work too!
- Badges
- Science topic
- Similar topics
- Analytical Chemistry
- Extraction