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Old 07-29-2010, 11:48 AM   #1
pmiguel
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Location: Purdue University, West Lafayette, Indiana

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Question RiboMinus yields

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...

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Old 01-23-2012, 06:46 AM   #2
mercier
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Default same results

Quote:
Originally Posted by pmiguel View Post
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
Hello I have exactly the same recovery... Did you improve this at this time??

Thank's
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Old 01-23-2012, 07:03 AM   #3
pmiguel
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We just never could get ribominus to work. So we switched to Epicentre Ribo-zero. To be fair, ribozero gave wildly variable results initially. But it looks like this was just the result of using an alcohol precipitation to concentrate the samples. When we switched to Zymo clean-up/concentration columns, things seemed to stabilize.

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Old 01-23-2012, 07:06 AM   #4
mercier
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Originally Posted by pmiguel View Post
We just never could get ribominus to work. So we switched to Epicentre Ribo-zero. To be fair, ribozero gave wildly variable results initially. But it looks like this was just the result of using an alcohol precipitation to concentrate the samples. When we switched to Zymo clean-up/concentration columns, things seemed to stabilize.

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Phillip
Thank you, I think I'will test Ribo-zero as soon as possible
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Old 06-21-2012, 07:28 AM   #5
Arvind Bhagwat
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Red face

New RiboZero Magnetic kit recommends purification (post rRNA removal) by a modified Qiagen RNeasy MiniElute. Here they recommend adding 2 x vol of EtOH than recommended by Qiagen. i.e. 100 ul -rRNA sample + 350 ul RLT buffer and then add 550 ul EtOH (instead of Qiagen recommended 250 ul).

Has anyone tried this? Does it really improve yields. Not aware of this I followed regular Qiagen protocol and I got 47, 63, 44, 32, 44, and 43 ng mRNA from 5 ug total RNA in my six sample reps, ~1% mRNA from total RNA.
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Old 06-21-2012, 08:01 AM   #6
pmiguel
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My guess is that you would just retain more of the low molecular weight RNA by adding more EtOH prior to binding on the column. But that is just a guess.

I would be sorely tempted to just use 1.8 volume of Ampure method instead.

What organism did the RNA come from? That would be a decent yield for a plant. As long as your concentration is correct. UV spec is strongly confounded by guanidine salts...

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Old 06-21-2012, 08:04 AM   #7
Arvind Bhagwat
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Organism is Salmonella, yields are measured using BioRad Expirion Hi sens chip.
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