The results are a little difficult to interpret. Initially for ten E. coli samples we started with 10 ug. After a single cycle of ribo-minus, the 23S and 16S rRNA looked to have been removed (by Bioanalyser pico-RNA chip analysis).
By UV-spectrophotometry (Nanodrop) it appeared we had what we considered quite good yields: 4-7.5%. However the Nanodrop spectra were questionable: there was a large peak -- probably remnant guanidinium from the "concentration modules" used post-depletion and most of the signal at 260 nm appeared to be a shoulder of that contaminant peak. Upon flurometric determination of the RNA concentration with Ribogreen we determined that our yields were actually 0.4%-1.2%.
Indeed this was less than the 200-500 ng recommended by the SOLiD total RNA seq kit when using ribo-depleted samples. Maddening.
I should add that we previously were seeing even lower yields from Ribo-minus. But I attribute this to substituting a heat block for a water bath in one of the steps. When we added water to the heat block well and allowed it to warm prior to the incubation (hence emulating a water bath) yields increased. Anecdotal, at best, since it had been months since our previous attempt with ribominus and that was with a eukaryote--but I can imagine there being a big difference between a water bath and a heat block.
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Phillip
By UV-spectrophotometry (Nanodrop) it appeared we had what we considered quite good yields: 4-7.5%. However the Nanodrop spectra were questionable: there was a large peak -- probably remnant guanidinium from the "concentration modules" used post-depletion and most of the signal at 260 nm appeared to be a shoulder of that contaminant peak. Upon flurometric determination of the RNA concentration with Ribogreen we determined that our yields were actually 0.4%-1.2%.
Indeed this was less than the 200-500 ng recommended by the SOLiD total RNA seq kit when using ribo-depleted samples. Maddening.
I should add that we previously were seeing even lower yields from Ribo-minus. But I attribute this to substituting a heat block for a water bath in one of the steps. When we added water to the heat block well and allowed it to warm prior to the incubation (hence emulating a water bath) yields increased. Anecdotal, at best, since it had been months since our previous attempt with ribominus and that was with a eukaryote--but I can imagine there being a big difference between a water bath and a heat block.
--
Phillip
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