Nonspecific RNA transcription assay - (Jun/26/2013 )

I'm optimizing a nonspecific RNA transcription assay to analyze purified RNA polymerase (RNAP). The assay involves incubating a sample with a reaction mix that contains unlabeled ATP, CTP, and GTP and P32 labeled UTP, stopping the reaction with 5% TCA, then loading the reaction onto glass filters, washing the filters, then measuring the radioactivity of each filters using a scintillation counter. I've optimized the protocol with commercial T7 RNAP and I'm able to get strong signal that is consistent between samples with equal amounts of RNAP. However, I'm now attempting to optimize the assay using E. coli cell lysate, but there is a huge variation between samples assaying the same volume of lysate. I'm not sure why there would be so much variation, since I don't see it with T7 RNAP. Has anyone used a similar nonspecific transcription assay and run into the same problem?

Was the density of the cell lysate the same in each case (i.e. did you grow it to the same OD)? Are there potential inhibitors in the medium or secreted from the bacteria?

I've been using the same lysate sample to optimize the assay, using 10 uL for each sample I want to run. The cells were around an OD of .9 when I harvested the cells.

I did a side by side comparison of lysate samples to which I added Rnase inhibitor and samples where I didn't, and there wasn't any difference in signal between them.

I've harvested fresh cells and resuspended them so that they're about twice as concentrated as before, I'll see if that makes a difference or not.

I tried some assays with the new lysate samples, and in order to determine if the increase in signal above the background is due to transcription, I boiled three 10 uL samples and compared that signal to lysate which was not boiled. I also boiled a sample containing commercial T7 polymerase to compare to a sample which was not boiled. The boiled T7 sample did not have signal above background, and the T7 I did not boil had a strong signal like I've seen before. However the signal from boiled lysate was not different from my sample which was not boiled, so whatever is causing the increase in signal is not actually due to polymerase activity.

I think the next thing I'm going to try is playing with the concentration of my labeled and unlabeled UTP. I did this when opitimizing the T7, but for cell lysate the optimal concentrations may be different.