Invitrogen RiboMinus. I thought we were getting roughly what the protocol specifies (>= 500 ng for 10 ug of input total RNA). But then after running an Agilent pico RNA chip on the rRNA-depleted RNA, it seemed we were getting closer to an order of magnitude less than that.
Yes, I know, Agilent labchip estimates of concentrations are not very accurate. So, we checked with a fluorimeter. The fluorimeter result was in the same ballpark as the labchip. Looking at the UV spectrum of the rRNA-depleted RNA, we see a hump at 250 nm that likely is the source of much of the 260 nm absorbance. Likely this is guanidine isothiocyanate -- a component of both the capture oligo binding buffer and the RNA "concentrator" silica columns binding reagent as well.
Is anyone getting good yields from Ribominus? (And is certain the yield is real, not a guanidine isothiocyanate artefact.) I would love to hear about it...
--
Phillip
Yes, I know, Agilent labchip estimates of concentrations are not very accurate. So, we checked with a fluorimeter. The fluorimeter result was in the same ballpark as the labchip. Looking at the UV spectrum of the rRNA-depleted RNA, we see a hump at 250 nm that likely is the source of much of the 260 nm absorbance. Likely this is guanidine isothiocyanate -- a component of both the capture oligo binding buffer and the RNA "concentrator" silica columns binding reagent as well.
Is anyone getting good yields from Ribominus? (And is certain the yield is real, not a guanidine isothiocyanate artefact.) I would love to hear about it...
--
Phillip
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