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Old 01-25-2012, 05:00 PM   #1
mateo
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Default tRNA/5S rRNA depletion

Dear all,
I'd like to deplete tRNA and 5S rRNA from total RNA to clone and deep sequence primarily precursor miRNAs in eukaryotic cells. I'm assuming that pre-miRNA are at a very low concentration when compared to tRNA/5S so if these species are left in my sample preparation they will represent the vast majority of sequences. Any help and/or suggestions would greatly be appreciated.
Thanks.
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Old 01-26-2012, 06:18 AM   #2
whw
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Gel purification can be a good way of eliminating larger species. I know that Bioo Scientific includes a gel purification step in their small RNA sequencing protocol.
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Old 01-26-2012, 08:32 AM   #3
pmiguel
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Quote:
Originally Posted by mateo View Post
Dear all,
I'd like to deplete tRNA and 5S rRNA from total RNA to clone and deep sequence primarily precursor miRNAs in eukaryotic cells. I'm assuming that pre-miRNA are at a very low concentration when compared to tRNA/5S so if these species are left in my sample preparation they will represent the vast majority of sequences. Any help and/or suggestions would greatly be appreciated.
Thanks.
Epicentre RiboZero Gold?

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Phillip
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Old 01-26-2012, 02:40 PM   #4
protist
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Quote:
Originally Posted by pmiguel View Post
Epicentre RiboZero Gold?

--
Phillip
RiboZero Gold will not remove tRNA, its advantage is it will remove mitochondrial as well nuclear encoded rRNA. To my knowledge aside from size selection of below tRNA size (~70 nt) there is not an effective kit out there..
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Old 01-26-2012, 02:48 PM   #5
mnkyboy
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You can see the tRNA pretty well in a gel. I would try the RZ beads to clean it up then gel purify.

Also it will probably be easier to purify out after library preparation than as RNA by gel.
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