Gene expression

[aftabac]

Hi
in my porject of cell culture, i want to moniter the expression of some genes at different stages, mean when they express more and at what stages less. so what are the best way to moniter the expression of genes at different stages. currently we donot have the facility of Real time PCR as well as Microarray, so excluding these please give some way to do this.

regards
aftab ahmad

[moleculardude]

Hey Aftab!

not sure exactly what you want to monitor as there are a myriad of ways to do this.

To monitor RNA expression of Gene of interest:

- check promoter activity using luciferase reporter gene linked to your promoter of interest. Transfect this into cells and examine expression of luciferase

- Real-time PCR and RT-PCR on cDNA can examine RNA expression as well
Northern Blot is also used sometimes

To Monitor Protein expression or Translation of a Gene of Interest:

- conduct western blot, immunoprecipiation or ELISA on your protein of interest. You need an antibody to do this.

- track the expression of the protein and localization of a GFP tagged protein. Use FRET also if you like.

- to measure translational rates, you could pulse radiolabel cells with radioactive methionine and check expression by autoradiography and antibody.

To more exactly measure translation, you could translate in vitro using cRNA and ribosomes. Use a reporter gene to track UTR translational rates.