How does 5EU click-chemistry-based nascent RNA isolation (as in Click-it nascent RNA capture kit from Thermo) compare with 4SU-based nascent RNA isolation in terms of replicability, sensitivity, and relative technical difficulty? My end goal is to compare the amount of RNA synthesized during a pulse of functionalized nucleotide to compare the amount of RNA produced from particular genes under different conditions. My end measurement is by RT-qPCR (or digital PCR if necessary). I have encountered large variations between technical replicates (harvesting identically treated cells) using the Thermo kit and am considering attempting the 4SU-based approach if it might be more reliable/user friendly (such as from the Dolken lab's protocol http://www.jove.com/video/50195/metabolic-labeling-newly-transcribed-rna-for-high-resolution-gene).
Hi Eric,
Did you manage to get the Click-it nascent RNA capture kit going? I am considering a project that involves capturing nascent RNA followed by higih throughput sequecing. Although you said you only used it for qPCR, I wanted to know if in the end it worked well for you.
Cheers,
Ariel
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