I am trying to overexpress a target gene in bovine adipocytes, i have clonned my target gene to pcDNA3.1 vector. There is 3 fold increase in the expression of target gene in the cells transfected with pcDNA+Terget gene as compared with blank cells (not transfected).
How much is the optimum fold increase in the expression of target gene through pcDNA vector for subsequent experement? Needs guidance please
Is this stable or transient transfection? If the former, are you assaying a pure clone, and if the latter, what is the transfection efficiency?
it is transient transfection,we hav,t checked transfection efficiency for pcDNA, however we checked transfection efficiency for siRNA and the same transfection protocol was followed for pcDNA
siRNA is quite different from plasmid DNA; not only is the chemical nature of the nucleic acid different (and depending on transfection methodology this does make a difference), but you use a vastly larger number of molecules in siRNA transfection. If I were you I would check transfection efficiency with a comparable plasmid (say, a GFP variant cloned into pcDNA3.1 and purified using the same kit), transfecting this control in parallel with your plasmid.
Hope it helps!
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Nucleic Acids
- RNA
- Small RNA
- microRNA