I have DNA extracted from human blood. I know that a certain protein has a variant and want to test if the expression level changes with the mutation and how much? As well as if there is any functional effects? And how can I approach them? This is the first time I am encountering a luciferase assay. I would like to test it in Hek293 cells, is it possible?
Can you clarify exactly what you're trying to do? I'm having trouble understanding your question fully. In my understanding, you are trying to figure out how a polymorphism affects the expression of that particular gene.
A luciferase assay won't test expression level of an mRNA or a protein, it will test the activity of a gene's promoter. If you want to see whether your mutation affects expression of the gene itself, you need to do real-time PCR and/or western blot (or some other method to authenticate protein expression).
If you're interested in finding out how a mutation of a transcription factor affects its ability to drive transcription, or how mutations on a gene promoter affect gene expression, the luciferase assay is a great technique.
Luciferase assay will help you in testing strength of any promoter, wild type or mutant. You need to clone promoter region in a certain vector such as pGL3 basic. There are kits from Promega to test the luciferase activity.
Thank you Kathleen and RIcha.
Would i be able to create a plasmid of the affected protein (genetic variant) and then test its expression with Luciferase?? Or by your response luciferase will only work for promoters??
In a luciferase assay, you will test only the activity of the promoter region. If the promoter region contains a genetic variant, you can definitely compare that promoter's activity to the promoter region of the wild-type gene. But from your question, I am getting the impression that your particular genetic variant includes a mutation in the coding sequence of the gene -- you wouldn't include the coding sequence in a plasmid created with a luciferase assay.
Here's an animation from Promega detailing how luciferase assays work: http://www.promega.com/resources/multimedia/reporter-assays-and-transfection/introduction-to-reporter-gene-assays/
- Badges
- Science method
- Similar topics
- Medicine
- Immunology
- Immunology Techniques
- Immunoassay
- ELISA