Reference RNA for qPCR - (Nov/26/2012 )
I am facing a problem regarding qPCR quantitation, and I hope, that someone in this forum might help me.
Currently I am working in a research project in which we perform a gene expression study of human monocytes. There is one group of critically ill patients, and another group which serves as a control group. However I want to perform both quantitation via deltadelta CT method in case PCR efficiencies are comparablem, but also quantitation via relative standard curves.
Now my question is: Which material is suitable for relative standard curve quantitation. I am not interested in absolute quantitation because I am comparing two groups together, but I am interested in how much mRNA content is in the cells independently regarding pcr efficiency.
Would it be appropriate to use THP-1 cells for reference rna?
Use control group pooled RNA as a calibrator for the diseased ones.
But also run all the control samples separately together with the pool to see the variation within group.
It's also possible to buy commercial reference human RNAs, but that's more if you want to create/fit in some universal reproducible ratio. In your case it's better to use your controls.
this sounds like a good Idea to me. Thank you very much.
I am wondering if there would be any benefit for the trial if I was performing a absolute quantitation with plasmids. I think to combine a relative quantitation with serial dilution for standard curve analysis from calibrator RNA and two endogenous reference genes should be fine to compare the gene expression levels between the two groups?