I have used the Thermo Fisher Click-IT Nascent RNA capture kit to capture the nascent transcripts and quantify them with qPCR, with no luck, either I get no count, the technical replicates are off, or I get artifacts.
Are there any assays, other than NRO, that can be done to quantify nascent transcripts?
Or is there a better NRO protocol that we can use that someone has had success with?
mRNA levels result from transcription but the stability of some of them are also tightly regulated.
Nuclear run-on assay is a great if not the best method to specifically evaluate elongating RNA resulting from transcription and not from a regulation of their stability.
I agree that nuclear run-on is a great if not the best way to quantify total nascent transcripts. But I think GRO-seq (developed in Dr. John Lis' lab at Cornell University) is the best way to get a picture of whole genome transcription activity, at the level of individual genes.
If you think about to measure just few nascent transcript you can prepare a simple RT-qPCR and design primer for intronic regions. Then you need to DNase treat your samples well and also use qPCR on the template with and without reverse transcription. Then you can eliminate the signal obtained from w/o from with RT.
And i forgot to mention that you have to use Trizol (or TRIreagent) for RNA isolation.
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