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Old 01-30-2014, 07:37 AM   #1
Neiltje
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Default RiboZero won't work?

Hi,

I'm trying to make a library of RNA following the ScriptSeq protocol. Everything works just fine except the RiboZero depletion of rRNA. I'm using young leafs of rice. An input of 2.5 g of sample, 8 l of removal solution.
I have to do this step twice and even then, I don't get rid of all the rRNA. I still have some nice peaks on the bioanalyzer.
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Old 02-03-2014, 10:18 AM   #2
kwaraska
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I know many kits have very specific maximums and just increasing the volumes doesn't always work.

I know Epicentre has a max of 1ug and over that causes incomplete reduction.

Not familiar with Script Seq but I would suggest checking the maximum amount and doing multiple replicates if you are over the maximum.
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Old 02-03-2014, 11:03 PM   #3
Neiltje
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Quote:
Originally Posted by kwaraska View Post
I know many kits have very specific maximums and just increasing the volumes doesn't always work.

I know Epicentre has a max of 1ug and over that causes incomplete reduction.

Not familiar with Script Seq but I would suggest checking the maximum amount and doing multiple replicates if you are over the maximum.

According to their protocol the input should be between 1 and 5 g. I'm using standard 2.5 g so that cannot be an issue
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Old 02-04-2014, 07:54 AM   #4
Olaf Blue
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Hi Neiltje, Which Ribo-Zero kit are you using, and what is the sample type (human/bacteria/yeast/plant)? RZ can use up to 5 g input total RNA, and depending upon the Ribo-Zero kit used, as little as 100 ng.
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Old 02-04-2014, 10:58 PM   #5
Neiltje
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Quote:
Originally Posted by Olaf Blue View Post
Hi Neiltje, Which Ribo-Zero kit are you using, and what is the sample type (human/bacteria/yeast/plant)? RZ can use up to 5 g input total RNA, and depending upon the Ribo-Zero kit used, as little as 100 ng.
I'm using the RiboZero plant leaf on young leafs of rice. I think it just doesn't work well on this material because the last time I tried, I also took a sample of Canola and that worked fine
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Old 02-05-2014, 01:59 PM   #6
Olaf Blue
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Variations in rRNA sequences even between some species can change the outcome. Do you have the rRNA sequences for your strain of rice?
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