Hi, Aramita. No, luciferase is not the only way. There are several reporter assay systems you can use.

Every system is going to have two essential pieces:

1) A promoter that is responsive (and specific) to the pathway you are studying.

2) A read-out that is easily measured.

I assume that you have a useful promoter, so allow me to address the second piece. You can quite easily find transcriptional reporters that utilize SEAP (Secreted Alkaline Phosphatase), beta-galactosidase (LacZ gene product), GFP (and variants), and RFP (and variants). You should choose the read-out based on the equipment you have available, and how precise the quantification needs to be. The other thing to consider is the half-life of the reporter protein itself -- the shorter the half-life (for instance, de-stabilized versions of GFP) the more accurate it's going to be with regard to temporal regulation... longer half-lives can mask regulation that changes over time.

Hope this helps,

Jonathan

Hi, Aramita. No, luciferase is not the only way. There are several reporter assay systems you can use.

Every system is going to have two essential pieces:

1) A promoter that is responsive (and specific) to the pathway you are studying.

2) A read-out that is easily measured.

I assume that you have a useful promoter, so allow me to address the second piece. You can quite easily find transcriptional reporters that utilize SEAP (Secreted Alkaline Phosphatase), beta-galactosidase (LacZ gene product), GFP (and variants), and RFP (and variants). You should choose the read-out based on the equipment you have available, and how precise the quantification needs to be. The other thing to consider is the half-life of the reporter protein itself -- the shorter the half-life (for instance, de-stabilized versions of GFP) the more accurate it's going to be with regard to temporal regulation... longer half-lives can mask regulation that changes over time.

Hope this helps,

Jonathan

Thanks Jonathan.

Well, then if I have an access to GFP assay kit, what I basically need is to have my GFP tagged promoter plasmid construct, right ???

Hi, Aramita.

I think I understand what you're asking -- if you're asking if you just need to generate a plasmid where GFP expression is under the control of your promoter, then yes, that's all you need.

If you're starting from the beginning, I'd recommend you try something like the rapid turnover systems by Clontech (http://www.clontech.com/US/Support/Applications/Using_Fluorescent_Proteins/EGFP_Vectors). Be sure to pick out one that is either promoter-less or has a minimal promoter that you can add your transcription factor binding sites to.

Feel free to contact me directly at [email protected] if you have any further questions.

Best wishes,

Jonathan

Hi Jonathan,

Thanks a lot for your suggestions. Will be implying them and I just hope to be successful. Your guidance may be needed and so a special thanks for your contact information.

If you are studying a promoter (promoter gene analysis) sure you could use reporter systems as suggested above but if you are looking at transcription activity of a gene a simple qRTPCR can suffice. Depending on what kind of scenario you are interested in treated as compared to non treated!!!.

@Edward : strictly speaking, monitoring RNA steady-state levels (e.g. by qRT-PCR) does not measure transcription, but the net amount of RNA, which is a combination of what is produced by transcription, and what is lost through degradation. Thus it can be indicative, but has to be confirmed at the transcription level.

Sure Vincent, a multiple of evaluation approaches are required to give a conclusive evaluation of active transcriptional activity. and yes mRNA stability, rate of depletion will surely bias a simple qRTPCR....My suggestion was on the basis of simplicity before getting caught-up in generating multiple promoter reporter tools which at the end of the day may not be relevant. Happened to me I went thru a series of cloning to generate promoter constructs that turned out to be non responsive to my treatments...Turns out that the response elements were located in the intron

If questions of stability may arise you could could as well use a nuclease protection assay