Alternatives to Nuclear Run-on?

[GWD]

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Northern Blot, RT-PCR, microarray analysis, etc.. all quantify steady-state mRNA levels.

I'm looking for an assay to evaluate changes in RATE of transcription.

Anyone know any alternate techniques to nuclear run-on?

 

[malchik]

How about measuring polII density at a locus by ChIP with anti-polII antibody followed by quantitative PCR?

 

[GWD]

How about measuring polII density at a locus by ChIP with anti-polII antibody followed by quantitative PCR?

that'd work... good call 👍

 

[StIGMA]

Would pulse labeling experiments accomplish what you're trying to do?

 

[ecoli]

Would pulse labeling experiments accomplish what you're trying to do?

you mean like florescence? I've heard of that being used in binding, perhaps you could adapt it to the binding of RNAP to the target sequence? I don't know enough about it to speculate further, however.

 

[StIGMA]

you mean like florescence? I've heard of that being used in binding, perhaps you could adapt it to the binding of RNAP to the target sequence? I don't know enough about it to speculate further, however.

With radiolabeled UTP.

 

[GWD]

you mean like florescence? I've heard of that being used in binding, perhaps you could adapt it to the binding of RNAP to the target sequence? I don't know enough about it to speculate further, however.

Nope.. Stigma's right.

Might work in your system, but eukaryotic polymerases are particuarly intolerent of flourescent nucleotide modifications.

 

[malchik]

After thinking about it, my ChIP suggestion probably wouldn't be acceptable to hardcore transcription people as a proxy for transcription rate. Mainly because it doesn't distinguish between initiation and elongation. Depends on how crucial the experiment is to your paper and on who reviews it. You may have to just do the run-on.

 

[GWD]

After thinking about it, my ChIP suggestion probably wouldn't be acceptable to hardcore transcription people as a proxy for transcription rate. Mainly because it doesn't distinguish between initiation and elongation. Depends on how crucial the experiment is to your paper and on who reviews it. You may have to just do the run-on.

Yeah.. I had thought of that.

I'm just sort of looking for ideas to throw out there though, so I think that's a viable option as long as I make it clear that I am aware of aforementioned caveat.

Essentially, I'm looking at differential protein expression in response to cytokine treatment. While you are absolutely correct that it doesn't distinguish between initiation and elongation, it's still a valid phenotypic change in response to dose.

But yeah.. if you can think of anything else that actually measures transcription rate, that would be cool too.

 

[ecoli]

perhaps you could do something simpler... like comparing mRNA expression levels (cDNA) of your target gene to a gene that has a continuous basal expression, like a housekeeping gene.

I've done this type of semi-quantitative analysis before, and it works well enough. I've never used it to measure the rate, but if you extract the mRNA and different time points and have a gene with a known transcription rate to compare it to, you might get a general idea, using a less technically challenging method.