I want to check the effect of an SNP (single nucleotide polymorphisms) on gene expression. For that I amplified two PCR fragments containing two different alleles. And then I ligated into PGL3 basic vector. I transfected my construct (1 µg) in U937 cell line by lipofectamine method. I used renilla (5 ng) as an internal control. I got quite high value of renilla in control PGL3 transfected cell line, whereas in constructs as well as in basic PGL3, the luciferase value was extremely low. What might be the possible reason for that?
Several factors could contribute to reduced luciferase (reporter) activity in the assay you performed. Because the reporter gene expression determines the levels of reporter activity, a PCR should allow you to determine whether the regulatory sequence has the ability to drive the expression of reporter gene RNA. If RNA is expressed, but luciferase activity is "extremely low" several approaches can be used to improve the activity, including increasing the amount of transfected DNA, length of incubation after transfection, reduction in cell lysis buffer volume etc.
This is a common problem with luciferase assays. What promoter is in your Renilla construct? It is likely that squelching is occurring in your experiments (pGL3 with a functional promoter competes for limiting factors that drive gene expression thus attenuating the activity of the promoter on the Renilla plasmid, of which there are relatively few copies. pGL3 basic alone will not have this squelching effect).
- Badges
- Science topic
- Similar topics
- Biological Science
- Genetics
- Gene Expression